Method of preparing an undifferentiated cell

ABSTRACT

Disclosed is a method of preparing an undifferentiated cell. The method includes contacting a more committed cell with an agent that causes the more committed cell to retrodifferentiate into an undifferentiated cell.

FIELD OF THE INVENTION

[0001] The present invention relates to a method of preparing anundifferentiated cell. In particular, the present invention relates to amethod of preparing an undifferentiated cell from a more committed cell.

[0002] In addition the present invention relates to the use of theundifferentiated cell of the present invention for the preparation of anew more committed cell—i.e. a recommitted cell.

[0003] The present invention also relates to the use of theundifferentiated cell of the present invention or the recommitted cellof the present invention to have an effect (directly or indirectly viathe use of products obtained therefrom) on the immune system, such asthe alleviation of symptoms associated with, or the partial or completecure from, an immunological condition or disease.

BACKGROUND TO THE INVENTION

[0004] Differentiation is a process whereby structures and functions ofcells are progressively committed to give rise to more specialisedcells, such as the formation of T cells or B cells from immaturehaemopoietic precursors. Therefore, as the cells become more committed,they become more specialised. In the majority of mammalian cell types,cell differentiation is a one-way process leading ultimately toterminally differentiated cells. However, although some cell typespersist throughout life without dividing and without being replaced,many cell types do continue to divide during the lifetime of theorganism and undergo renewal. This may be by simple division (e.g. livercells) or, as in the case of cells such as haemopoietic cells andepidermal cells, by division of relatively undifferentiated stem cellsfollowed by commitment of one of the daughter cells to a programme ofsubsequent irreversible differentiation. All of these processes,however, have one feature in common: cells either maintain their stateof differentiation or become more differentiated. They do not becomeundifferentiated or even less differentiated.

[0005] Retrodifferentiation is a process whereby structures andfunctions of cells are progressively changed to give rise to lessspecialised cells. Some cells naturally undergo limited reversedifferentiation (retrodifferentiation) in vivo in response to tissuedamage. For example, liver cells have been observed to revert to anenzyme expression pattern similar to the foetal enzymic pattern duringliver regeneration (Curtin and Snell, 1983, Br. J. Cancer, Vol 48;495-505).

[0006] Jose Uriel (Cancer Research, 1976, vol 36, pp 4269-4275)presented a review on the topic of retrodifferentiation, in which hesaid:

[0007] “retrodifferentiation appears as a common adaptive process forthe maintenance of cell integrity against deleterious agents of variedetiology (physical, chemical, and viral). While preserving the entireinformation encoded on its genome, cells undergoing retrodifferentiationlose morphological and functional complexity by virtue of a process ofself-deletion of cytoplasmic structures and the transition to a morejuvenile pattern of gene expression. This results in a progressiveuniformization of originally distinct cell phenotypes and to a decreaseof responsiveness to regulatory signals operational in adult cells.Retrodifferentiation is normally counterbalanced by a process ofreontogeny that tends to restore the terminal phenotypes where thereversion started. This explains why retrodifferentiation remainsinvariably associated to cell regeneration and tissue repair.”

[0008] Uriel (ibid) then went on to discuss cases of reportedretrodifferentiation—such as the work of Gurdon relating to nuclei fromgut epithelial cells of Xenopus tadpoles (Advances in Morphogenesis,1966, vol 4, pp 1-43. New York Academic Press, Eds Abercrombie andBracher), and the work of Bresnick relating to regeneration of liver(Methods in Cancer Research, 1971, vol 6, pp 347-391).

[0009] Uriel (ibid) also reported on work relating to isolated liverparenchymal cells for in vitro cultures. According to Uriel:

[0010] “Contrary to the results with fetal or neonatal hepatocytes, withhepatocytes from regenerating liver, or from established hepatomas, ithas been difficult to obtain permanent class lines from resting adulthepatocytes.”

[0011] Uriel (ibid) also reported on apparent retrodifferentiation incancer, wherein he stated:

[0012] “the biochemical phenotypes of many tumours show analogouschanges of reversion toward immaturity . . . during the preneoplasticphase of liver carcinogenesis, cells also retrodifferentiate.”

[0013] More recent findings on retrodifferentiation include the work ofMinoru Fukunda (Cancer Research, 1981, vol 41: 4621-4628). Fukundainduced specific changes in the cell surface glycoprotein profile ofK562 human leukaemic cells by use of the tumour-promoting phorbol ester,12-O-tetradecanoyl-phorbol- 13-acetate (TPA). According to Fukunda TPAappeared to induce the K562 human leukaemic cells into aretrodifferentiated stage.

[0014] Also, Hass et al. (Cell Growth & Differentiation, 1991, vol 2:541-548) reported that long term culture of TPA-differentiated U-937leukaemia cells in the absence of phorbol ester for 32-36 days resultedin a process of retrodifferentiation and that the retrodifferentiatedcells detached from the substrate and reinitiated proliferation.

[0015] As mentioned above, another reported case of retrodifferentiationis the work of Curtin and Snell (Br. J. Cancer, 1983, vol 48: 495-505).These workers compared enzymatic changes occurring duringdiethylnitrosamine-induced hepatocarcinogenesis and liver regenerationafter partial hepatectomy to normal liver differentiation. Thesesworkers found changes in enzyme activities during carcinogenesis thatwere similar to a step-wise reversal of differentiation. According tothese workers, their results suggest that an underlyingretrodifferentiation process is common to both the process ofhepatocarcinogenesis and liver regeneration.

[0016] More recently, Chastre et al. (FEBS Letters, 1985, vol 188 (2),pp2810-2811) reported on the retrodifferentiation of the human coloniccancerous subclone HT29- 18.

[0017] Even more recently, Kobayashi et al. (Leukaemia Research, 1994,18 (12): 929-933) have reported on the establishment of aretrodifferentiated cell line (RD-1) from a single ratmyelomonocyticleukemia cell which differentiated into a macrophage-likecell by treatment with lipopolysaccharide (LPS).

[0018] Much of the above prior art focuses on retrodifferentiation as astage in carcinogenesis. Several prior art documents refer toexperiments where tumour cell lines have apparently beenretrodifferentiated. However, these prior art experiments were carriedout using tumour cell lines. The situation in genetically aberranttumour cell lines is not comparable with the normal differentiationpathways. Indeed, it is questionable whether these results indicate trueretrodifferentiation in the sense of normal cell lineages. Further, thevast majority of the prior art retrodifferentiated cells were incapableof redifferentiating to a more committed cell, whether of the samelineage, or of any other lineage. One exception is given in Kobayashi etal., 1994, Vol 18; 929-933 where a retrodifferentiated tumour cell linewas differentiated into a macrophage-like cell using lipopolysaccharide.However, the retrodifferentiation achieved was very limited and thecells remained committed both before and after treatment.

[0019] Similarly, the reverse differentiation seen to occur naturally inliver cells is also very limited and can more accurately be classed asmodulations of the differentiated state, that is to say, reversiblechanges between closely related cell phenotypes.

SUMMARY OF THE INVENTION

[0020] Contrary to all earlier teachings, we have now shown that it ispossible to treat differentiated cells so that they becomeundifferentiated cells, including stem cells. These undifferentiatedcells are capable of proliferating and giving rise to redifferentiatedprogeny of the same lineage or any other lineage. We believe that theprocess responsible for these changes is retrodifferentiation and thuswe have now surprisingly found that it is possible to reverse thedifferentiation process in normal differentiated cells obtained from thehuman patients to produce a stem cell. Furthermore, in the case ofretrodifferentiated haematopoietic cells, these stem cells arepluripotent and can give rise to more than one cell lineage.

[0021] The clinical implications of this finding are enormous. Stemcells are extremely difficult to obtain from human patients. They aretypically obtained from umbilical tissue, bone marrow or blood wherethey are present in only very small amounts. However, the presentinvention provides a method for producing stem cells from more committedcells by the process of retrodifferentiation. Since more committed cells(such as B lymphocytes) are much more abundant in the human body, thistechnique provides a powerful new method for obtaining stem cells. Thehaemopoietic stem cells exemplified in the present invention arepluripotent and are therefore capable of redifferentiating along morethan one cell lineage

[0022] Thus, according to a first aspect of the present invention thereis provided a method of preparing an undifferentiated cell, the methodcomprising contacting a more committed cell with an agent that causesthe more committed cell to retrodifferentiate into an undifferentiatedcell.

[0023] In a specific embodiment there is provided a method of increasingthe relative number of undifferentiated cells in a cell populationincluding committed cells, which method comprises:

[0024] (i) contacting the cell population with an agent that operablyengages said committed cells; and

[0025] (ii) incubating committed cells that are engaged by said agentsuch that the relative number of undifferentiated cells increases as aresult of said engaging.

[0026] Preferably, the agent engages a receptor that mediates capture,recognition or presentation of an antigen at the surface of thecommitted cells. More preferably, the receptor is an MHC class I antigenor an MHC class II antigen, such as a class I antigen selected fromHuman-Leukocyte-Associated (HLA) -A receptor, an HLA-B receptor, anHLA-C receptor, an HLA-E receptor, an HLA-F receptor or an HLA-Greceptor or a class II antigen selected from an HLA-DM receptor, anHLA-DP receptor, an HLA-DQ receptor or an HLA-DR receptor.

[0027] Typically, the committed cells are differentiated cells,preferably cells selected from T-cell colony-forming cells (CFC-Tcells), B-cell colony-forming cells (CFC-B cells), eosinophilcolony-forming cells (CFC-Eosin cells), basophil colony-forming cells(CFC-Bas cells), granulocyte/monocyte colony-forming cells (CFC-GMcells), megakaryocyte colony-forming cells (CFC-MEG cells), erythrocyteburst-forming cells (BFC-E cells), erythrocyte colony-forming cells(CFC-E cells), T cells and B cells.

[0028] In one preferred embodiment of the present invention, the morecommitted cell is not a cancer cell. In another preferred embodiment ofthe present invention, the agent is neither carcinogenic nor capable ofpromoting cancer growth.

[0029] In a preferred embodiment, the agent an antibody to the receptor,such as a monoclonal antibody to the receptor. Specific examples includeCR3/43 and monoclonal antibody TAL. 1B5.

[0030] Preferably the agent is used in conjunction with a biologicalresponse modifier, such as an alkylating agent, for example alkylatingagent that is or comprises cyclophosphoamide.

[0031] Preferred undifferentiated cells comprises a stem cell antigen.In a preferred embodiment, the undifferentiated cells are selected froman embryonic stem cell, a pluripotent stem cell, a lymphoid stem celland a myeloid stem cell. Preferably, the undifferentiated cells arecharacterised by one or more of following cell surface markerdesignations: CD34⁺, HLA-DR⁻, CD38⁻ and/or CD45low. More preferably theundifferentiated cell is CD34⁺ and CD38⁻, even more preferably, CD34⁺,CD38⁻, HLA-DR⁻ and CD45low.

[0032] Thus in a preferred embodiment the present invention alsoprovides a method of increasing the relative number of cells having acell surface marker designation CD34⁺, CD38⁻, HLA-DR⁻ and/or CD45low ina cell population including committed cells, which method comprises:

[0033] (i) contacting the cell population with an agent that operablyengages said committed cells; and

[0034] (ii) incubating committed cells that are engaged by said agentsuch that the relative number of CD34⁺, HLA-DR⁻ and/or CD45low cellsincreases as a result of said engaging.

[0035] In another embodiment, the present invention provides a method ofinducing committed cells in a cell population to become undifferentiatedcells capable of being recommitted into more differentiated cells whichmethod comprises:

[0036] (i) contacting the cell population with an agent that operablyengages said committed cells; and

[0037] (ii) incubating committed cells that are engaged by said agentsuch that they become undifferentiated cells as a result of saidengaging.

[0038] In a further embodiment, the present invention provides a methodof producing an altered cell population comprising increased numbersundifferentiated cells capable of being recommitted into moredifferentiated cells, which method comprises:

[0039] (i) contacting an initial cell population comprising committedcells with an agent that operably engages said committed cells; and

[0040] (ii) incubating committed cells that are engaged by said agentsuch that they become undifferentiated cells as a result of saidengaging, thereby resulting in an altered cell population comprisingincreased numbers of said undifferentiated cells.

[0041] In any of the above methods, an optional step (iii) of enrichingsaid undifferentiated cells or recovering said undifferentiated cellsfrom the altered cell population may be performed. Preferably, step(iii) comprises enriching said undifferentiated cells or recovering saidundifferentiated cells from the altered cell population by using a cellsurface marker present on the cell surface of the undifferentiated cellor a cell surface marker present on the surface of the committed cellsbut substantially absent from the cell surface of the undifferentiatedcells. Examples of suitable markers include CD34, CD45 and HLA-DR.

[0042] In another preferred embodiment, the undifferentiated cell of theinvention is CD34⁻ CD45⁻ and negative for markers of haemopoeiticlineages.

[0043] The undifferentiated cells produced by the methods of the presentinvention may be subsequently redifferentiated. Accordingly, the presentinvention provides a method of producing a committed/more differentiatedcell which method comprises contacting an undifferentiated cell producedby the methods of the invention with a compound that stimulatesdifferentiation of the undifferentiated cell. Suitable compounds includegrowth factors, colony stimulating factors and cytokines.

[0044] Thus according to a second aspect of the present invention thereis provided a method comprising contacting a more committed cell with anagent that causes the more committed cell to retrodifferentiate into anundifferentiated cell; and then committing the undifferentiated cell toa recommitted cell.

[0045] The term “recommitted cell” means a cell derived from theundifferentiated cell—i.e. a new more committed cell. “More committed”means more differentiated and can easily be determined by reference toknown pathways and stages of cell differentiation.

[0046] According to a third aspect of the present invention there isprovided an undifferentiated cell produced according to the method ofthe present invention.

[0047] According to a fourth aspect of the present invention there isprovided an undifferentiated cell produced according to the method ofthe present invention as or in the preparation of a medicament.

[0048] According to a fifth aspect of the present invention there isprovided a recommitted cell produced according to the method of thepresent invention.

[0049] The more differentiated cells may be of the same lineage as theoriginal committed cells or of different lineage.

[0050] Thus as well as producing undifferentiated cells, the methods ofthe present invention can be used to convert cells of one lineage tothose of another lineage. Accordingly, in a further aspect the presentinvention provides a method of inducing in a cell population comprisingcommitted hemopoietic cells of one hemopoietic lineage to become cellsof another hemopoietic lineage which method comprises:

[0051] (i) contacting the cell population with an agent that engages areceptor that mediates capture, recognition or presentation of anantigen at the surface of said committed hemopoietic cells; and

[0052] (ii) incubating committed hemopoietic cells that are engaged bysaid agent such that they become cells of another hemopoietic lineage asa result of said engaging.

[0053] Preferably said committed cells are of a B cell lineage andbecome cells of another hemopoietic lineage selected from a T celllineage and a myeloid lineage.

[0054] Undifferentiated cells produced according to the methods of thepresent invention may be used to manufacture a medicaments for thetreatment of an immunological disorder or disease. Similarly,recommitted cells produced according to the methods of the presentinvention may be used to manufacture a medicaments for the treatment ofan immunological disorder or disease.

[0055] Thus, in its broadest sense, the present invention is based onthe highly surprising finding that it is possible to form anundifferentiated cell from a more committed cell.

[0056] The present invention is highly advantageous as it is nowpossible to prepare undifferentiated cells from more committed cells andthen use those undifferentiated cells as, or to prepare, medicamentseither in vitro or in vivo or combinations thereof for the treatments ofdisorders.

[0057] The present invention is also advantageous as it is possible tocommit the undifferentiated cell prepared by retrodifferentiation to arecommitted cell, such as a new differentiated cell, with a view tocorrecting or removing the original more committed cell or forcorrecting or removing a product thereof.

[0058] Preferably, the more committed cell is capable ofretrodifferentiating into an MHC Class I⁺ and/or an MHC Class II⁺undifferentiated cell.

[0059] Preferably, the more committed cell is capable ofretrodifferentiating into an undifferentiated cell comprising a stemcell antigen.

[0060] Preferably, the more committed cell is capable ofretrodifferentiating into a CD34⁺ undifferentiated cell.

[0061] Preferably, the more committed cell is capable ofretrodifferentiating into a lymphohaematopoietic progenitor cell.

[0062] Preferably, the more committed cell is capable ofretrodifferentiating into a pluripotent stem cell.

[0063] The findings presented herein may also be used to identifyfurther agents that are capable of effecting retrodifferentiation ofcommitted cells to undifferentiated cells. Accordingly, the presentinvention provides a method for identifying a substance capable ofretrodifferentiating a committed/differentiated cell to anundifferentiated cell, which method comprises contacting a population ofcells comprising committed cells with a candidate substance anddetermining whether there is an increase in the relative numbers ofundifferentiated cells in said cell population.

[0064] Preferably, said increase occurs within 24 hours, preferably 4 to8 hours (such that any changes cannot be solely accounted for by cellproliferation).

[0065] Typically, the determination of changes in the numbers ofundifferentiated cells is performed by monitoring changes in the numbersof cell having cell surface markers characteristic of undifferentiatedcells. Examples of suitable cell surface markers include CD34⁺.Alternatively, or in addition, decreases in the numbers of cells havingcell surface markers typical of differentiated cells and notundifferentiated cells may be monitored.

[0066] Preferably the committed cells used in the assay are committedhemopoietic cells such as cells selected from CFC-T cells, CFC-B cells,CFC-Eosin cells, CFC-Bas cells, CFC-GM cells, CFC-MEG cells, BFC-Ecells, CFC-E cells, T cells and B cells, more preferably B cells.

[0067] The present invention also provides an agent identified by theassay method of the invention and its use in a retrodifferentiationmethod of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0068]FIG. 1 depicts various haemopoietic cells

[0069]FIG. 2 is a scheme depicting differentiation pathways fromlymphoid stem cells.

[0070]FIG. 3 is a scheme depicting differentiation pathways from myeloidstem cells.

[0071]FIG. 4 is a diagram of lymphohaemopoietic progenitor cells.

[0072]FIG. 5 is a scatter graph showing flow cytometry results.

[0073]FIG. 6 is a microscope picture of cells before treatment accordingto the method of the present invention.

[0074]FIG. 7 is a microscope picture of cells prepared by the method ofthe present invention.

[0075]FIG. 8 is a microscope picture of cells prepared by the method ofthe present invention but at a lower magnification.

[0076]FIG. 9 is a microscope picture of cells before treatment accordingto the method of the present invention.

[0077]FIG. 10 is a microscope picture of cells prepared by the method ofthe present invention.

[0078]FIG. 11 is a microscope picture of cells prepared by the method ofthe present invention.

[0079]FIG. 12 is a photomicrograph of a blood sample from a BCLL patientbefore treatment according to the method of the present invention. A andB are at different magnifications.

[0080]FIG. 13 is a photomicrograph of a blood sample from a BCLL patientduring, treatment according to the method of the present invention

[0081]FIG. 14 is a photomicrograph of a blood sample from a BCLL patientduring treatment according to the method of the present invention

[0082]FIG. 15 is a photomicrograph of a blood sample from a BCLL patientafter treatment according to the method of the present invention,showing cells prepared by the method of the invention.

[0083]FIG. 16 is a photograph of a Southern blot.

[0084]FIG. 17 is (A) a photograph of an agarose gel containing PCRproducts resolved by electrophoresis and stained with ethidium bromideand (B) a photograph of a Southern blot.

[0085]FIG. 18 depicts scatter graphs showing flow cytometry results forhealthy cells treated with one of three different agents.

[0086]FIG. 19 is a photomicrograph showing the results of a colonyforming assay conducted using purified normal B cells treated accordingto the methods of the invention.

[0087]FIG. 20 is a photomicrograph, using inverted bright fieldmicroscopy, showing the establishment of a long-term culture of stemcells (undifferentiated) produced according to the present invention (3days following treatment).

[0088]FIG. 21 is a confocal microscopy image of cells before and aftertreatment according to the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0089] I. Undifferentiated Cells and Differentiated Cell

[0090] There are many undifferentiated cells and differentiated cellsfound in vivo and the general art is replete with general teachings onthem.

[0091] By way of example, with respect to cells of the haempoietic celllineages, reference may be made to inter alia Levitt and Mertelsman 1995(Haematopoietic Stem Cells, published by Marcel Dekker Inc—especiallypages 45-59) and Roitt et al. (Immunology, 4th Edition, Eds. Roitt,Brostoff and Male 1996, Publ. Mosby—especially Chapter 10).

[0092] An undifferentiated cell is an immature cell that does notdisplay a mature differentiated character but is capable of yieldingprogeny that do. A well-known example of an undifferentiated cell is astem cell.

[0093] Stem cells are undifferentiated immature cells, capable of selfrenewal (division without limit) and differentiation (specialisation).These juvenile cells are abundant in a developing embryo, however, theirnumbers decrease as development progresses. By contrast, an adultorganism contain limited number of stem cells which are confined tocertain body compartments.

[0094] It is generally believed that stem cells are either monopotent,bipotent or pluripotent. Monopotent and bipotent stem cells are morerestricted in development and give rise to one or two types ofspecialised cells, respectively. In contrast, the pluripotent stem cells(PSCs) can differentiate into many different types of cells, giving riseto tissue (which constitute organs) or in the case of totipotent stemcells, the whole organism.

[0095] Pluripotent stem cells, unlike monopotent or bipotent, arecapable of multilineage differentiation, giving rise to a tissue whichwould consist of a collection of cells of different types or lineages.

[0096] According to the current understanding, as borne out by theteachings found on page 911 of Molecular Biology of the Cell (pub.Garland Publishers Inc. 1983) and more recently Levitt and Mertelsman(ibid), a stem cell, such as a pluripotent stem cell, has the followingfour characteristics:

[0097] i. it is an undifferentiated cell—i.e. it is not terminallydifferentiated;

[0098] ii. it has the ability to divide without limit;

[0099] iii. it has the ability to give rise to differentiated progeny;and

[0100] iv. when it divides each daughter has a choice: it can eitherremain as stem cell like its parent or it can embark on a course leadingirreversibly to terminal differentiation.

[0101] Note should be made of the last qualification, namely thataccording to the general teachings in the art once an undifferentiatedcell has differentiated to a more committed cell it can not thenretrodifferentiate. This understanding was even supported by theteachings of Uriel (ibid), Fukunda (ibid), Hass et al (ibid), Curtin andSnell (ibid), Chastre et al (ibid), and Kobayashi et al (ibid) as theseworkers retrodifferentiated certain types of differentiated cells butwherein those cells remained committed to the same lineage and they didnot retrodifferentiate into undifferentiated cells.

[0102] Therefore, according to the state of the art before the presentinvention, it was believed that it was not possible to formundifferentiated cells, such as stem cells, from more committed cells.However, the present invention shows that this belief is inaccurate andthat it is possible to form undifferentiated cells from more committedcells.

[0103] The Haematopoietic Stem Cell is an example of a pluripotent stemcell which is found among marrow cells and gives rise to all the variousblood cells (including leucocytes and erythrocytes).

[0104] Blood is a fluid tissue, consisting of Lymphocyte (Ly), Monocytes(Mo), Neutrophils (Ne), Basophils (Ba), Eosinophils (Eso), Platelets(PI) and Red Blood Cells (Rbc)—see FIG. 1. This specialised tissue isproduced by the differentiation of Haematopoietic Stem Cells (Hsc). Ingeneral, the white blood cells (inside blue circle) fight infectionswhile red blood cells (inside green circle) transport nutrients, oxygenand waste product around the body.

[0105] Previously, haemopoietic stem cells were extracted by isolationfrom (i) bone marrow, (ii) growth factor mobilised peripheral blood or(iii) cord blood (placenta). Recently, haemopoietic stem cells have beenprepared from Embryonic Stem Cells, which are extracted from embryosobtained using in vitro fertilisation techniques. These undifferentiatedcells are capable of multi-lineage differentiation and reconstitution ofall body tissue i.e. are totipotent.

[0106] The above mentioned extraction methods are cumbersome, sometimehazardous and in certain instances can be argued unethical, especially,in the case of the Embryonic Stem Cells extraction method.

[0107] There are a number of undifferentiated stem cells of thehaemopoietic lineage. These include pluripotent stem cells (PSCs),lymphoid stem cells (LSCs) and myeloid stem cells (MSCs), knowncollectively as lymphohaematopoietic progenitor cells (LPCs). LSCs andMSCs are each formed by the differentiation of PSCs. Hence, LSCs andMSCs are more committed than PSCs.

[0108] Examples of differentiated cells of the haemopoietic lineageinclude T cells, B cells, eosinophils, basophils, neutrophils,megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, andlymphocytes.

[0109] T cells and B cells are formed by the differentiation of LSCs.Hence, T cells and B cells are more committed than LSCs. In more detail,the chain of differentiation is LSC→pro-B-cell or prothymocyte.Pro-B-cell→pre-B-cell→mature B-cell→plasma cell. Prothymocyte→commonthymocyte→mature thymocytes (helper/inducer or cytotoxic/suppressorlineages)—see FIG. 2.

[0110] Eosinophils, basophils, neutrophils, megakaryocytes, monocytes,erythrocytes, granulocytes, mast cells, NKs, and lymphocytes are formedby the differentiation of MSCs. Hence, each of these cells are morecommitted than MSCs. In more detail, the chain of differentiation isMSC→immature megakaryoblast (→megakaryoblast→megakaryocyte→platelet) orproerythroblast (→erythroblast→reticulocyte→erythrocyte) ormyelomonocytic stem cell, a bipotent stem cell that differentiates toeither a myeloblast (→promyelocyt→myelocyt→granulocyte) or a monoblast(→promonocyte→monocyte→macrophage)—see FIG. 3.

[0111] The pathways of differentiation of haemotopoiesis have thus beenextensively characterised and the various cell stages are readilyidentifiable according to morphology and lineage-specific cell surfacemarkers (see below).

[0112] Other stem cells include neural stem cells, multipotent stemcells that can generate neurons, atrocytes and oligodendrocytes(Nakafuku and Nakamura, 1995, J. Neurosci Res., vol 41(2): 153-68;Anderson, 1994, FASEB J., vol 8(10): 707-13; Morshead et al., 1994,Neuron, Vol 13(5): 1071-82). Skeletal muscle satellite cells are anothertype of stem cell, more specifically a distinct class of myogenic cellsthat are maintained as quiescent stem cells in the adult and can giverise to new muscle cells when needed (Bischoff, 1986, Dev Biol., vol115(1): 129-39). Other types of stem cells are epithelial stem cells, asubset of basal cells, and mesenchymal stem cells.

[0113] A very important type of stem cells are embryonic stem (ES)cells. These cells have been extensively studied and characterised.Indeed, ES cells are routinely used in the production of transgenicanimals. ES cells have been shown to differentiate in vitro into severalcell types including lymphoid precursors (Potocnik et al., 1994, EMBOJ., vol 13(22): 5274-83) and neural cells. ES cells are characterised bya number of stage-specific markers such as stage-specific embryonicmarkers 3 and 4 (SSEA-3 and SSEA-4), high molecular weight glycoproteinsTRA-1-60 and TRA-1-81 and alkaline phosphatase (Andrews et al., 1984,Hybridoma, vol 3: 347-361; Kannagi et al., 1983, EMBO J., vol 2:2355-2361; Fox et al., 1984, Dev. Biol., vol 103: 263-266; Ozawa et al.,1985, Cell. Differ., vol 16: 169-173).

[0114] Various antigens are associated with undifferentiated anddifferentiated cells. The term “associated” here means the cellsexpressing or capable of expressing, or presenting or capable of beinginduced to present, or comprising, the respective antigen(s).

[0115] Most undifferentiated cells and differentiated cells compriseMajor Histocompatability Complex (MHC) Class I antigens and/or Class IIantigens. If these antigens are associated with those cells then theyare called Class I⁺ and/or Class II⁺ cells.

[0116] Each specific antigen associated with an undifferentiated cell ora differentiated cell can act as a marker. Hence, different types ofcells can be distinguished from each other on the basis of theirassociated particular antigen(s) or on the basis of a particularcombination of associated antigens.

[0117] Examples of these marker antigens include the antigens CD34, CD19and CD3. If these antigens are present then these particular cells arecalled CD34⁺, CD19⁺ and CD3⁺ cells respectively. If these antigens arenot present then these cells are called CD34⁻, CD19⁻ and CD3⁻ cellsrespectively.

[0118] In more detail, PSCs are CD34⁺ DR⁻ TdT⁻ cells (other usefulmarkers being CD38⁻ and CD36⁺). LSCs are DR⁺, CD34⁺ and TdT⁺ cells (alsoCD38⁺). MSCs are CD34⁺, DR⁺, CD13⁺, CD33⁺, CD7⁺ and TdT⁺ cells. B cellsare CD19⁺, CD21⁺, CD22⁺ and DR⁺ cells. T cells are CD2⁺, CD3⁺, andeither CD4⁺ or CD8⁺ cells. Immature lymphocytes are CD4⁺ and CD8⁺ cells.Activated T cells are DR⁺ cells. Natural killer cells (NKs) are CD56⁺and CD16⁺ cells. T lymphocytes are CD7⁺ cells. Leukocytes are CD45⁺cells. Granulocytes are CD13⁺ and CD33⁺ cells. Monocyte macrophage cellsare CD14⁺ and DR⁺ cells. Additional details are provided in FIGS. 2 and3.

[0119] Embryonic stem cells express SSEA-3 and SSEA-4, high molecularweight glycoproteins TRA-1-60 and TRA-1-81 and alkaline phosphatase.They also do not express SSEA-1, the presence of which is an indicatorof differentiation. Other markers are known for other types of stemcells, such as Nestein for neuroepithelial stem cells (J. Neurosci,1985, Vol 5: 3310). Mesenchymal stem cells are positive for SH2, SH3,CD29, CD44, CD71, CD90, CD106, CD120a and CD124, for example, andnegative for CD34, CD45 and CD14.

[0120] Alternatively, or in addition, many cells can be identified bymorphological characteristics. The identification of cells usingmicroscopy, optionally with staining techniques is an extremely welldeveloped branch of science termed histology and the relevant skills arewidely possessed in the art. Clearly staining of cells will only becarried out on aliquots of cells to confirm identity since stains ingeneral cause cell death.

[0121] Hence, by looking for the presence of the above-listed antigenmarkers it is possible to identify certain cell types (e.g. whether ornot a cell is an undifferentiated cell or a differentiated cell) and thespecialisation of that cell type (e.g. whether that cell is a T cell ora B cell).

[0122] Undifferentiated cells may comprise any components that areconcerned with antigen presentation, capture or recognition. Preferably,the undifferentiated cell is an MHC Class I⁺ and/or an MHC Class II⁺cell.

[0123] The more committed cell may comprise any components that areconcerned with antigen presentation, capture or recognition. Preferably,the more committed cell is an MHC Class I⁺ and/or an MHC Class II⁺ cell.

[0124] The more committed cell is any cell derived or derivable from anundifferentiated cell. Thus, in one preferred embodiment, the morecommitted cell is also an undifferentiated cell. By way of exampletherefore the more committed undifferentiated cell can be a lymphoidstem cell or a myeloid stem cell, and the undifferentiated cell is apluripotent stem cell.

[0125] In another preferred embodiment, the more committed cell is adifferentiated cell, such as a CFC-T cell, a CFC-B cell, a CFC-Eosincell, a CFC-Bas cell, a CFC-Bas cell, a CFC-GM cell, a CFC-MEG cell, aBFC-E cell, a CFC-E cell, a T cell, a B cell, an eosinophil, a basophil,a neutrophil, a monocyte, a megakaryocyte or an erythrocyte; and theundifferentiated cell is a myeloid stem cell, a lymphoid stem cell or apluripotent stem cell.

[0126] If the more committed cell is a differentiated cell thenpreferably the differentiated cell is a B lymphocyte (activated ornon-activated), a T lymphocyte (activated or non-activated), a cell fromthe macrophage monocyte lineage, a nucleated cell capable of expressingclass I or class II antigens, a cell that can be induced to expressclass I or class II antigens or an enucleated cell (i.e. a cell thatdoes not contain a nucleus—such as a red blood cell).

[0127] In alternative preferred embodiments, the differentiated cell isselected from any one of a group of cells comprising large granularlymphocytes, null lymphocytes and natural killer cells, each expressingthe CD56 and/or CD16 cell surface receptors.

[0128] The differentiated cell may even be formed by the nucleation ofan enucleated cell.

[0129] II. Agents

[0130] The agent operably engages the more committed cell in order toretrodifferentiate that cell into an undifferentiated cell. In thisregard, the agent for the retrodifferentiation of the more committedcell into the undifferentiated cell may act in direct engagement or inindirect engagement with the more committed cell.

[0131] The agent may act intracellularly within the more committed cell.However, preferably, the agent acts extracellularly of the morecommitted cell.

[0132] An example of direct engagement is when the more committed cellhas at least one cell surface receptor on its cell surface, such as aβ-chain having homologous regions (regions that are commonly foundhaving the same or a similar sequence) such as those that may be foundon B cells, and wherein the agent directly engages the cell surfacereceptor. Another example, is when the more committed cell has a cellsurface receptor on its cell surface such as an α-chain havinghomologous regions such as those that may be found on T cells, andwherein the agent directly engages the cell surface receptor.

[0133] An example of indirect engagement is when the more committed cellhas at least two cell surface receptors on its cell surface andengagement of the agent with one of the receptors affects the otherreceptor which then induces retrodifferentiation of the more committedcell.

[0134] The agent for the retrodifferentiation of the more committed cellinto an undifferentiated cell may be a chemical compound or composition.Preferably, however, the agent is capable of engaging a cell surfacereceptor on the surface of the more committed cell. Thus, in a preferredembodiment, the agent operably engages a receptor present on the surfaceof the more committed cell—which receptor may be expressed by the morecommitted cell, such as a receptor that is capable of being expressed bythe more committed cell.

[0135] For example, preferred agents include any one or more of cyclicadenosine monophosphate (cAMP), a CD4 molecule, a CD8 molecule, a partor all of a T-cell receptor, a ligand (fixed or free), a peptide, aT-cell receptor (TCR), an antibody, a cross-reactive antibody, amonoclonal antibody, or a polyclonal antibody. Growth factors may alsobe used, such as haemopoietic growth factors, for example erythropoietinand granulocyte-monocyte colony stimulating factor (GM-CSF).

[0136] If the agent is an antibody, a cross-reactive antibody, amonoclonal antibody, or a polyclonal antibody, then preferably the agentis any one or more of an antibody, a cross-reactive antibody, amonoclonal antibody, or a polyclonal antibody to any one or more of: theβ chain of a MHC class II antigen, the β chain of a MHC HLA-DR antigen,the α chain of a MHC class I or class II antigen, the α chain of HLA-DRantigen, the α and the β chain of MHC class II antigen or of a MHC classI antigen. An example of a suitable antibody is CR3/43 (supplied byDako).

[0137] The term “antibody” includes the various fragments (whetherderived by proteolytic cleavage or recombinant technology) andderivatives that retain binding activity, such as Fab, F(ab′)₂ and scFvantibodies, as well as mimetics or bioisosteres thereof. Also includedas antibodies are genetically engineered variants where some of theamino acid sequences have been modified, for example by replacement ofamino acid residues to enhance binding or, where the antibodies havebeen made in a different species to the organism whose cells it isdesired to treat according to the methods of the invention, to decreasethe possibility of adverse immune reactions (an example of this is‘humanised’ mouse monoclonal antibodies).

[0138] Agents used to effect the conversion of a more committed cell toan undifferentiated cell preferably act extracellularly of the morecommitted cell. In particular, it is preferred that the more committedcell comprises a receptor that is operably engageable by the agent andthe agent operably engages the receptor.

[0139] For example the receptor may be a cell surface receptor. Specificexamples of cell surface receptors include MHC class I and class IIreceptors. Preferably, the receptor comprises an α-component and/or aβ-component, as is the case for MHC class I and class II receptors.

[0140] More preferably, the receptor comprises a β-chain havinghomologous regions, for example at least the homologous regions of theβ-chain of HLA-DR.

[0141] Alternatively, or in addition, the receptor comprises an α-chainhaving homologous regions, for example at least the homologous regionsof the α-chain of HLA-DR.

[0142] Preferably, the receptor is a Class I or a Class II antigen ofthe major histocompatibility complex (MHC). In preferred embodiments thecell surface receptor is any one of: an HLA-DR receptor, a DM receptor,a DP receptor, a DQ receptor, an HLA-A receptor, an HLA-B receptor, anHLA-C receptor, an HLA-E receptor, an HLA-F receptor, or an HLA-Greceptor. In more preferred embodiments the cell surface receptor is anHLA-DR receptor.

[0143] Preferably, the agent is an antibody to the receptor, morepreferably the agent is a monoclonal antibody to the receptor.

[0144] Another preferred example of an agent is one that modulates MHCgene expression such as MHC Class I⁺ and/or MHC Class II⁺ expression.

[0145] In a preferred embodiment, the agent is used in conjunction witha biological response modifier. Examples of biological responsemodifiers include an alkylating agent, an immunomodulator, a growthfactor, a cytokine, a cell surface receptor, a hormone, a nucleic acid,a nucleotide sequence, an antigen or a peptide. A preferred alkylatingagent is or comprises cyclophosphoamide.

[0146] Other preferred biological response modifiers include compoundscapable of upregulating MHC class I and/or class II antigen expression.In a preferred embodiment, this is so as to allow an agent that binds toan MHC receptor to work more effectively. Since any cell type can bemade to express MHC class I and/or class II antigens, this shouldprovide a method for retrodifferentiation a wide variety of cell typeswhether they constitutively express class I and/or class II MHC antigensor not.

[0147] III. Methods for Retrodifferentiating Cells

[0148] In the methods of the invention, a population of cells comprisingcommitted cells is contacted with an agent that operably engages one ormore committed cell in the population. The cell population is thenincubated so as to allow those cells that have been operably engaged bythe agent to progress through the retrodifferentiation process andultimately become undifferentiated.

[0149] Preferably the contacting step comprises the agent engaging withany one or more of the following: homologous regions of the α-chain ofclass I antigens, homologous regions of the α-chain of class IIantigens, a CD4 cell surface receptor, a CD8 cell surface receptor,homologous regions of the β-chain of class II antigens in the presenceof lymphocytes, homologous regions of the α-chain of class I antigens inthe presence of lymphocytes, or homologous regions of the α-chain ofclass II antigens in the presence of lymphocytes. Preferably thecontacting step occurs in the presence of the biological responsemodifier (see above).

[0150] Typically, the population of cells is derived from a biologicalsample, such as blood or related tissues including bone marrow, neuronaltissue from the central nervous system or peripheral nervous system, ormuscle tissue. Preferably biological material is of post-natal origin.It is preferred to use whole blood or processed products thereof, suchas plasma, since their removal from subjects can be carried out with theminimum of medical supervision. Blood samples are typically treated withanticoagulents such as heparin or citrate. Cells in the biologicalsample may be treated to enrich certain cell types, remove certain celltypes or dissociate cells from a tissue mass. Useful methods forpurifying and separating cells include centrifugation (such as densitygradient centrifugation), flow cytometry and affinity chromatography(such as the use of magnetic beads comprising monoclonal antibodies tocell surface markers or panning). By way of example, Ficoll-Hypaqueseparation is useful for removing erythrocytes and granulocytes to leavemononuclear cells such as lymphocytes and monocytes.

[0151] Since the cells are essentially primary cultures, it maynecessary to supplement populations of cells with suitable nutrients tomaintain viability. Suitable culture conditions are known by the skilledperson in the art. Nonetheless, treatment of cell populations ispreferably initiated as soon as possible after removal of biologicalsamples from patients, typically within 12 hours, preferably within 2 to4 hours. Cell viability can be checked using well known techniques suchas trypan blue exclusion.

[0152] Cell populations are generally incubated with an agent for atleast two hours, typically between 2 and 24 hours, preferably between 2and 12 hours. Incubations are typically performed at from about roomtemperature, for example about 22° C., up to about 37° C. including 33°C. The progress of the retrodifferentiation procedure can be checkedperiodically by removing a small aliquot of the sample and examiningcells using microscopy and/or flow cytometry.

[0153] Once the relative numbers of the desired cell type have increasedto a suitable level, which may for example be as low as 0.1% or as highas 5%, the resulting altered cell populations may be used in a number ofways. With respect to the numbers of undifferentiated cells formed, itis important to appreciate the proliferative ability of stem cells.Although under some circumstance, the numbers of stem cells or otherundifferentiated cells formed may appear to be low, studies have shownthat only 50 pluripotent haemopoietic stem cells can reconstitute anentire haemopoietic system in a donor mouse. Thus therapeutic utilitydoes not require the formation of a large number of cells by the methodsof the invention.

[0154] Conversion of more committed cells to undifferentiated cells mayalso be carried out in vivo by administration of the agent, admixed witha pharmaceutically carrier or diluent, to a patient. However it ispreferred in many cases that retrodifferentiation is performed invitro/ex vivo.

[0155] Treated populations of cells obtained in vitro may be usedsubsequently with minimal processing. For example they may be simplycombined with a pharmaceutically acceptable carrier or diluent andadministered to a patient in need of stem cells.

[0156] It may however be desirable to enrich the cell population for theundifferentiated cells or purify the cells from the cell population.This can conveniently be performed using a number of methods. Forexample cells may be purified on the basis of cell surface markers usingchromatography and/or flow cytometry. Nonetheless, it will often beneither necessary nor desirable to extensively purify undifferentiatedcells from the cell population since other cells present in thepopulation (for example stromal cells) may maintain stem cell viabilityand function.

[0157] Flow cytometry is a well-established, reliable and powerfultechnique for characterizing cells within mixed populations as well asfor sorting cells. Flow cytometry operates on the basis of physicalcharacteristics of particles in liquid suspension, which can bedistinguished when interrogated with a beam of light. Such particles mayof course be cells. Physical characteristics include cell size andstructure or, as has become very popular in recent years, cell surfacemarkers bound by monoclonal antibodies conjugated to fluorescentmolecules.

[0158] Kreisseg et al., 1994, J. Hematother 3(4): 263-89, state,“Because of the availability of anti-CD34 monoclonal antibodies,multiparameter flow cytometry has become the tool of choice fordetermination of haemapoietic stem and progenitor cells” and goes on todescribe general techniques for quantitation and characterisation ofCD34-expressing cells by flow cytometry. Further, Korbling et al., 1994,Bone Marrow Transplant. 13: 649-54, teaches purification of CD34⁺ cellsby immunoadsorption followed by flow cytometry based on HLA-DRexpression. As discussed above, CD34⁺ is a useful marker in connectionwith stem cells/progenitor cells.

[0159] Flow cytometry techniques for sorting stem cells based on otherphysical characteristics are also available. For example, Visser et al.,1980, Blood Cells 6:391-407 teach that stem cells may be isolated on thebasis of their size and degree of structuredness. Grogan et al., 1980,Blood Cells, 6: 625-44 also teach that “viable stem cells may be sortedfrom simple haemapoietic tissues in high and verifiable purity”.

[0160] As well as selecting for cells on the basis of the presence of acell surface marker or other physical property (positive selection),cell populations may be enriched, purified using negative criteria. Forexample, cells that possess lineage specific markers such as CD4, CD8,CD42 and CD3 may be removed from the cell population by flow cytometryor affinity chromatography.

[0161] A very useful technique for purifying cells involves the use ofantibodies or other affinity ligands linked to magnetic beads. The beadsare incubated with the cell population and cells that have a cellsurface marker, such as CD34, to which the affinity ligand binds arecaptured. The sample tube containing the cells is placed in a magneticsample concentrator where the beads are attracted to the sides of thetube. After one or more wash stages, the cells of interest have beenpartially or substantially completely purified from other cells. Whenused in a negative selection format, instead of washing cells bound tothe beads by discarding the liquid phase, the liquid phase is kept andconsequently, the cells bound to the beads are effectively removed fromthe cell population.

[0162] These affinity ligand-based purification methods can be used withany cell type for which suitable markers have been characterized or maybe characterized.

[0163] Urbankova et al., 1996. (J. Chromatogr B Biomed Appl. 687:449-52) teaches the micropreparation of hemopoietic stem cells from amouse bone marrow suspension by gravitational field-flow fractionation.Urbankova et al., 1996, further comments that the method was used forthe chacterization of stem cells from mouse bone marrow because thesecells are bigger than the other cells in bone marrow and it is thereforepossible to separate them from the mixture. Thus physical parametersother than cell surface markers may be used to purify/enrich for stemcells.

[0164] Cell populations comprising undifferentiated cells and purifiedundifferentiated cells produced by the methods of the invention may bemaintained in vitro using known techniques. Typically, minimal growthmedia such as Hanks, RPMI 1640, Dulbecco's Miminal Essential Media(DMEM) or Iscove's Modified Dulbecco Medium are used, supplemented withmammalian serum such as FBS, and optionally autologous plasma, toprovide a suitable growth environment for the cells. In a preferredembodiment, stem cells are cultured on feeder layers such as layers ofstromal cells (see Deryugina et al., 1993, Crit Rev. Immunology, vol 13:115-150). Stromal cells are believed to secrete factors that maintainprogenitor cells in an undifferentiated state. A long term culturesystem for stem cells is described by Dexter et al., 1977 (J. CellPhysiol, vol 91: 335) and Dexter et al., 1979 (Acta. Haematol., vol 62:299).

[0165] For instance, Lebkowski et al., 1992 (Transplantation 53(5):1011-9) teaches that human CD34⁺ haemopoietic cells can be purifiedusing a technology based on the use of monoclonal antibodies that arecovalently immobilised on polystyrene surfaces and that the CD34⁺ cellspurified by this process can be maintained with greater than 85%viability. Lebkowski et al., 1993 (J. Hematother, 2(3): 339-42) alsoteaches how to isolate and culture human CD34⁺ cells. See also Haylocket al., 1994 (Immunomethods, vol 5(3): 217-25) for a review of variousmethods.

[0166] Confirmation of stem cell identity can be performed using anumber of in vitro assays such as CFC assays (see also, the examples).Very primitive haemopoietic stem cells are often measured using thelong-term culture initiating cell (LTC-IC) assay (Eaves et al, 1991, J.Tiss. Cult. Meth. Vol 13: 55-62). LTC-ICs sustain haemopoiesis for 5 to12 weeks.

[0167] Cell populations comprising undifferentiated cells and purifiedpreparations of comprising undifferentiated cells may be frozen forfuture use. Suitable techniques for freezing cells and subsequentlyreviving them are known in the art.

[0168] IV. Methods for Recommitting Undifferentiated Cells

[0169] One important application of undifferentiated cells of thepresent invention is in the reconstitution of tissues, for examplenervous tissue or haemopoietic cells. This involves differentiating theundifferentiated cells produced by the methods of the invention. Thismay be carried out by simply administering the undifferentiated cells toa patient, typically at a specific site of interest such as the bonemarrow or spinal cord, and allowing the natural physiological conditionswithin the patient to effect differentiation. A specific example of thisis the reconstitution or supplementation of the haemopoietic system, forexample in the case of AIDS patients with reduced number of CD4⁺lymphocytes.

[0170] Alternatively, differentiation (also termed “recommitting”,herein) can be effected in vitro and expanded cells then, for example,administered therapeutically. This is generally performed byadministering growth factors. For example, retinoic acid has been usedto differentiate ES cells into neuronal cells. Methylcellulose followedby co-culture with a bone marrow stromal line and IL-7 has been used todifferentiate ES cells into lymphocyte precursors (Nisitani et al.,1994, Int. Immuno., vol 6(6): 909-916). Bischoff, 1986 (Dev. Biol., vol115(1): 129-39) teaches how to differentiate muscle satellite cells intomature muscle fibres. Neural precursor cells can be expanded with basicfibroblast growth factor and epidermal growth factor (Nakafuku andNakamura, 1995, J. Neurosci. Res., vol 41(2): 153-168). Haemopoieticstem cells can be expanded using a number of growth factors includingGM-CSF, erythropoeitin, stem cell factor and interleukins (IL-1, IL-3,IL-6)—see Metcalf, 1989 (Nature, vol 339: 27-30) for a review of thesevarious factors.

[0171] Potocnik et al., 1994 (EMBO J., vol 13(22): 5274-83) evendemonstrated the differentiation of ES cells to haemopoietic cells usinglow oxygen (5%) conditions.

[0172] Thus, in a preferred embodiment of the present invention theundifferentiated cell is then committed into a recommitted cell, such asa differentiated cell. The recommitted cell may be of the same lineageto the more committed cell from which the undifferentiated cell wasderived. Alternatively, the recommitted cell may be of a differentlineage to the more committed cell from which the undifferentiated cellwas derived. For example, a B lymphocyte may be retrodifferentiated to aCD34⁺ CD38⁻ HLA-DR⁻ stem cell. The stem cell may be subsequentlyrecommitted along a B cell lineage (the same lineage) or a lymphoidlineage (different lineage).

[0173] V. Assays for Identifying Retrodifferentiating Agents

[0174] In addition to the agents mentioned above, further suitableagents may be identified using assay methods of the invention. Thus, thepresent invention provides a method for identifying a substance capableof retrodifferentiating a committed/differentiated cell to anundifferentiated cell, which method comprises contacting a population ofcells comprising committed cells with a candidate substance anddetermining whether there is an increase in the relative numbers ofundifferentiated cells in said cell population.

[0175] Suitable candidate substances include ligands that bind to cellsurface receptors such as antibody products (for example, monoclonal andpolyclonal antibodies, single chain antibodies, chimeric antibodies andCDR-grafted antibodies), such as antibodies that bind to cell surfacereceptors. Cell surface receptors of particular interest are describedabove and include MHC receptors and surface proteins of with CDdesignations, such as CD4 and CD8. Other ligands that bind to cellsurface receptors include growth factors.

[0176] Furthermore, combinatorial libraries, peptide and peptidemimetics, defined chemical entities, oligonucleotides, and naturalproduct libraries may be screened for activity as retrodifferentiationagents. The candidate substances may be used in an initial screen inbatches of, for example 10 substances per reaction, and the substancesof those batches which show inhibition tested individually.

[0177] A typical assay comprises placing an aliquot of cells comprisingcommitted cells in a suitable vessel such as a multiwell plate. Acandidate substance is added to the well and the cells incubated in thewell. Incubations are typically performed at from about roomtemperature, for example about 22° C., up to about 37° C. including 33°C.

[0178] Retrodifferentiation may be measured by removing a small aliquotof cells and examining the cells by microscopy and/or flow cytometry todetermine whether there has been a change in the numbers ofundifferentiated cells. Typically, the determination of changes in thenumbers of undifferentiated cells is performed by monitoring changes inthe numbers of cell having cell surface markers characteristic ofundifferentiated cells, although morphological changes may also be usedas a guide. Examples of suitable cell surface markers include CD34⁺.Alternatively, or in addition, decreases in the numbers of cells havingcell surface markers typical of differentiated cells and notundifferentiated cells may be monitored, for example a reduction in therelative numbers of cells possessing lineage specific markers such asCD3, CD4 and CD8

[0179] Preferably, any increase in the numbers of cells havingcharacteristics typical of undifferentiated cells occurs within 24hours, preferably 4 to 8 hours, such that any changes cannot be solelyaccounted for by cell proliferation.

[0180] It may be desirable to prescreen for agents that bind to, forexample, cell surface receptors, such as MHC class I or class IIreceptors. Any agents identified as binding to target cell surfacereceptors may then be used in the above assay to determine their effecton retrodifferentiation. As a particular example, phage displaylibraries which express antibody binding domains may be used to identifyantibody fragments (typically scFvs) that bind to a target cell surfacemarker, such as the homologous region of the β-chain of MHC class IIreceptors. Suitable binding assays are known in the art, as is thegeneration and screening of phage display libraries. Assays may also beused to identify optimised antibodies or antibody fragments, for exampleto screen a mutagenised library of derivatives of an antibody alreadyshown to effect retrodifferentiation.

[0181] VI. Uses

[0182] The present invention provides methods of retrodifferentiatingcommitted cells to undifferentiated cells. In particular, the presentinvention provides a method for preparing a stem cell from a moredifferentiated cell. The clinical implications of this are enormoussince stem cells are being used in a wide variety of therapeuticapplications but up until now were difficult, cumbersome and sometimesethically controversial to obtain.

[0183] Stem cells produced according to the present invention may beused to repopulate specific cell populations in a patient, such as ahaemopoietic cell population or a subpopulation thereof, such as CD4T-lymphocytes. The more committed cells used to produce the stem cellsmay be from the same patient or a matched donor. Thus stem cellsproduced according to the present invention may be used to heal andreconstitute specialised cell tissue and organs.

[0184] Thus, the present invention also encompasses a medicamentcomprising an undifferentiated cell prepared by any one of theseprocesses admixed with a suitable diluent, carrier or excipient.

[0185] In one embodiment, the medicament comprising the undifferentiatedcell may be used produce a beneficial more committed cell, such as onehaving a correct genomic structure, in order to alleviate any symptomsor conditions brought on by or associated with a more committed cellhaving an incorrect genomic structure. Thus, the present invention alsoprovides a process of removing an acquired mutation from a morecommitted cell wherein the method comprises forming an undifferentiatedcell by the method according to the present invention, committing theundifferentiated cell into a recommitted cell, whereby arrangement orrearrangement of the genome and/or nucleus of the cell causes themutation to be removed.

[0186] Preferably the gene is inserted into the immunoglobulin region orTCR region of the genome.

[0187] The present invention also provides a method of treating apatient suffering from a disease or a disorder resulting from adefective cell or an unwanted cell, the method comprising preparing anundifferentiated cell by contacting a more committed cell with an agentthat causes the more committed cell to retrodifferentiate into theundifferentiated cell, and then optionally committing theundifferentiated cell into a recommitted cell; wherein theundifferentiated cell, or the recommitted cell, affects the defectivecell or the unwanted cell to alleviate the symptoms of the disease ordisorder or to cure the patient of the disease or condition.

[0188] Alternatively, the undifferentiated cell could be used to producea more committed cell that produces an entity that cures any symptoms orconditions brought on by or associated with a more committed cell havingan incorrect genomic structure.

[0189] For example, the present invention may be used to prepareantibodies or T cell receptors to an antigen that is expressed by themore committed cell which has retrodifferentiated into theundifferentiated cell. In this regard, the antigen may be a fetospecificantigen or a cross-reactive fetospecific antigen.

[0190] The present invention also includes a process of controlling thelevels of undifferentiated cells and more committed cells. For example,the present invention includes a method comprising forming anundifferentiated cell by the method according to the present inventionand then activating an apoptosis gene to affect the undifferentiatedcell, such as bring about the death thereof.

[0191] In a preferred embodiment the present invention relates to aprocess of introducing a gene into the genome of an undifferentiatedcell, wherein the process comprises introducing the gene into a morecommitted cell, and then preparing an undifferentiated cell by themethod according to the present invention, whereby the gene is presentin the undifferentiated cell.

[0192] In a more preferred embodiment the present invention relates to aprocess of introducing a gene into the genome of an undifferentiatedcell, wherein the process comprises inserting the gene into the genomeof a more committed cell, and then preparing an undifferentiated cell bythe method according to the present invention, whereby the gene ispresent in the undifferentiated cell.

[0193] The gene may be a gene that renders the undifferentiated cell andmore differentiated cells obtained therefrom more resistant topathogenic infections such as a viral infection. In particular, by wayof example, B lymphocytes from AIDS patients may be used to produce stemcells that are then engineered to be resistant to HIV infection. Whenexpanded and introduced into the patients, the resulting helper Tlymphocytes may also be resistant to HIV infection.

[0194] In an alternative embodiment the present invention relates to aprocess of introducing a gene into an undifferentiated cell, wherein theprocess comprises inserting the gene into the genome of a more committedcell, and then preparing an undifferentiated cell by the methodaccording to the present invention, whereby the gene is present in thegenome of the undifferentiated cell.

[0195] In addition, the present invention also encompasses the method ofthe present invention for preparing an undifferentiated cell, whereinthe method includes committing the undifferentiated cell into arecommitted cell and then fusing the recommitted cell to a myeloma. Thisallows the expression in vitro of large amounts of the desired product,such as an antibody or an antigen or a hormone etc.

[0196] The present invention encompasses an undifferentiated cellprepared by any one of these processes of the present invention.

[0197] Other aspects of the present invention include:

[0198] The use of any one of the agents of the present invention forpreparing an undifferentiated cell from a more committed cell.

[0199] The use of an undifferentiated cell produced according to themethod of the present invention for producing any one of a monoclonal ora polyclonal or a specific antibody from a B-lymphocyte or aT-lymphocyte; a cell from the macrophage monocyte lineage; a nucleatedcell capable of expressing class I or class II antigens; a cell capableof being induced to express class I or class II antigens; an enucleatedcell; a fragmented cell; or an apoptic cell.

[0200] The use of an undifferentiated cell produced according to themethod of the present invention for producing effector T-lymphocytesfrom B-lymphocytes and/or vice versa.

[0201] The use of an undifferentiated cell produced according to themethod of the present invention for producing any one or more of: amedicament, such as a medicament comprising or made from a B-lymphocyte,a T-lymphocyte, a cell from the macrophage monocyte lineage, a nucleatedcell capable of expressing a class I or a class II antigen, a cellcapable of being induced to express a class I or a class II antigen, oran enucleated cell.

[0202] The present invention also encompasses processes utilising theafore-mentioned uses and products or compositions prepared from suchprocesses.

[0203] The present invention also encompasses a medicament comprising anundifferentiated cell according to the present invention or a productobtained therefrom admixed with a suitable diluent, carrier orexcipient.

[0204] In one preferred embodiment the medicament comprises an antibodyor antigen obtained from an undifferentiated cell according to thepresent invention admixed with a suitable diluent, carrier or excipient.

[0205] Preferably the medicament is for the treatment of any one of:cancer, autoimmune diseases, blood disorders, cellular or tissueregeneration, organ regeneration, the treatment of organ or tissuetransplants, or congenital metabolic disorders.

[0206] The methods of the invention and products obtained by thosemethods, such as undifferentiated cells, may be used in research, forexample to study retrodifferentiation, differentiation and identify andstudy new developmental antigens and cluster differentiation antigens.

[0207] VII. Administration

[0208] Stem cells and recommitted cells of the present invention, aswell as agents shown to retrodifferentiate cells, may be used intherapeutic methods. Preferably the cells or agents of the invention arecombined with various components to produce compositions of theinvention. More preferably the compositions are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition (which may be for human or animal use).Suitable carriers and diluents include isotonic saline solutions, forexample phosphate-buffered saline. The composition of the invention maybe administered by direct injection. The composition may be formulatedfor parenteral, intramuscular, intravenous, subcutaneous, intraocular,oral or transdermal administration.

[0209] Compositions comprising cells are typically delivered byinjection or implantation. Cells may be delivered in suspension orembedded in a support matrix such as natural and/or syntheticbiodegradable matrices. Natural matrices include collagen matrices.Synthetic biodegradable matrices include polyanhydrides and polylacticacid. These matrices provide support for fragile cells in vivo and arepreferred for non-haemopoetic cells.

[0210] Delivery may also be by controlled delivery i.e. over a period oftime which may be from several minutes to several hours or days.Delivery may be systemic (for example by intravenous injection) ordirected to a particular site of interest.

[0211] Cells are typically administered in doses of from 1×10⁵ to 1×10⁷cells per kg. For example a 70 kg patient may be administered 14×10⁶CD34⁺ cells for reconstitution of haemopoietic tissues.

[0212] The routes of administration and dosages described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage for anyparticular patient and condition.

[0213] The present invention will now be described by way of examples,which are illustrative only and non-limiting.

[0214] A. Materials and Methods

[0215] Patients

[0216] Blood samples were obtained in lavender top tubes containing EDTAfrom patients with B-cell chronic lymphocytic leukaemias, patients withantibody deficiency (including IgA deficiency and X-linked infantilehypogammaglobulinaemias), patients with HIV infections and AIDSsyndrome, a patient with CMV infection, a patient with Hodgkin'slymphomas, a patient with acute T-cell leukaemia, a 6-days old baby withblastcytosis, various patients with various infections and clinicalconditions, cord blood, bone marrow's, and enriched B-lymphocytepreparations of healthy blood donors.

[0217] Clinical and Experimental Conditions

[0218] The clinical and experimental treatment conditions of patients,including various types of treatment applied to their blood samples, aredescribed in Table 1. Differential white blood cell (WBC) counts wereobtained using a Coulter Counter and these are included in the sameTable.

[0219] Treatment of Blood

[0220] Blood samples, once obtained, were treated with pure monoclonalantibody to the homologous region of the β-chain of the HLA-DR antigen(DAKO) and left to mix on a head to head roller at room temperature fora maximum of 24 hours. Some samples were mixed first on a head to headroller for 15 minutes after which they were left to incubate in anincubator at 22° C. The concentration of monoclonal antibody added toblood samples varied from 10-50 μl/ml of blood.

[0221] In addition, other treatments treatments were applied at the sameconcentrations and these included addition of a monoclonal antibody tothe homologous of the α-chain of the HLA-DR antigen, a monoclonalantibody to the homologous region of class I antigens, a monoclonalantibody to CD4, a monoclonal antibody to CD8, and a PE conjugatedmonoclonal antibody to the homologous region of the β-chain of theHLA-DR antigen.

[0222] Other treatments included the simultaneous addition of monoclonalantibodies to the homologous regions of the α and β-chains of the HLA-DRantigen to blood samples.

[0223] Furthermore, alkylating agents such as cyclophosphoamide wereadded to blood samples in combination with pure monoclonal antibody tothe homologous region of the β-chain of the HLA-DR antigen.

[0224] Following these treatments blood samples were stained with panelsof labelled monoclonal antibodies as instructed by the manufacturer'sinstructions and then analyzed using flow cytometry.

[0225] Incubation periods with monoclonal antibodies ranged from 2 hour,4 hour, 6 hour, 12 hour to 24 hour intervals.

[0226] Labelled Antibodies

[0227] The following monoclonal antibodies were used to detect thefollowing markers on cells by flow cytometry: CD19 and CD3, CD4 and CD8,DR and CD3, CD56 & 16 and CD3, CD45 and CD14, CD8 and CD3, CD8 and CD28,simultest control (IgG1 FITC+IgG2a PE), CD34 and CD2, CD7 and CD13 & 33,CD10 and CD25, CD5 and CD10, CD5 and CD21, CD7 and CD5, CD13 and CD20,CD23 and CD57 and CD25 and CD45 RA (Becton & Dickenson and DAKO).

[0228] Each patient's blood sample, both treated and untreated, wasanalyzed using the majority of the above panel in order to account forthe immunophenotypic changes that accompanied different types oftreatments and these were carried out separately on different aliquotsof the same blood sample. Untreated samples and other control treatmentswere stained and analyzed simultaneously.

[0229] Flow Cytometry

[0230] Whole blood sample was stained and lysed according to themanufacturer instructions. Flow cytomery analysis was performed on aFACScan@ with either simultest or PAINT A GATE software (BDIS) whichincluded negative controls back tracking. 10,000 to 20,000 events wereacquired and stored in list mode files.

[0231] Morphology

[0232] Morphology was analyzed using microscopy and Wright's stain.

[0233] Preparing Stem Cells from Enriched or Purified B-CLL (or Normal)Lymphocytes:

[0234] Aseptic techniques should be used throughout the followingprocedures:

[0235] (A) Mononuclear Cell Separation:

[0236] (i) Obtain mononuclear cells from peripheral blood samples bycentrifugation on Histopaque, Lymphoprep, or any Lymphocyte separationmedium (sp. grav 1.077) for 30 mins at 400 g.

[0237] (ii) Collect mononuclear cells in a 50 ml conical tube and washwith 30 mls of Hank's balanced salt solution (Ca²⁺ and Mg⁺ free, Sigma)containing 2% FCS and 2 mM EDTA or 0.6% citrate.

[0238] (iii) After washing, count cells and assess viability usingtrypan blue and haemocytometer.

[0239] (iv) If B-cell count is high, above 70% (20×10⁹/L, WBC), proceedstraight to B (vi).

[0240] (v) If B-cell count is low, below 70% (20×10⁹/L, WBC). Performnegative selection using Macs microbeads or FacsVantage purificationtechnique, as described below in Section 1. C.

[0241] (vi) Resuspend cell pellet at a concentration of 3×10⁶/ml in IMDMmedium (100 μg/ml streptomycin), containing 10% FCS (heat inactivated)and 10% HS (heat inactivated). Note: If no FCS and HS available, use 20%to 50% autologous plasma.

[0242] (B) Cell Treatment Using Pure CR3/43 (Dako) Monoclonal Antibody:

[0243] After mononuclear cell separation has been achieved in A (vi),proceed with the following:

[0244] (i) Use a culture tray with six wells, add 2 mls of cellsuspension [from A (vi) above] to each well of this multi-well culturetray.

[0245] (ii) Treat five wells each with 7.5 μl/ml of CR3/43 -(puremonoclonal antibody, Dako) and leave one well untreated (negativecontrol.

[0246] (iii) Incubate the culture tray in 5% CO₂ at 37 C.°.

[0247] (C) Purification of Cells:

[0248] Negative Selection of B cells using MACS microbeads (MiltenyiBiotec, here it is best to follow manufacturer instructions):

[0249] (i) Obtain mononuclear cells as in Section A above.

[0250] (ii) Pellet and resuspend cells in a final volume of 300 μl per10⁸ total cells in HBSS (consisting of 2% FCS and 2 mM EDTA or 0.6%citrate).

[0251] (iii) Add 100 μl per 10⁸ total cells of pure monoclonal antibodyto CD2 (IgG1, DAKO).

[0252] (iv) To the same cell suspension add 50 μl per 10⁸ per totalcells of pure monoclonal antibody to CD33 (IgG1, DAKO).

[0253] (v) Leave the mixture to incubate for 10 minutes at roomtemperature.

[0254] (vi) Wash cells with HBSS (containing 2% FCS and 2 mM EDTA) andresuspend at a final concentration of 400 μl per 10⁸ total cells, withthe same buffer.

[0255] (vii) Add 100 μl of rabbit anti-mouse IgG1 labelled microbeadsper 10⁸ total cells (or follow manufacturer instructions).

[0256] (viii) Thoroughly mix cells and incubate at 6 C.° to 12 C.°(fridge) for 15 minutes.

[0257] (ix) Again wash cells with HBSS (containing 2% FCS and 2 mM EDTA)and resuspend at a final concentration of 500 μl per 10⁸ total cells,with the same buffer.

[0258] (x) Assemble MS+/RS+ column in the magnetic field of the MACSseparator.

[0259] (xi) Wash column with 3 mls of HBSS (containing 2% FCS and 2 mMEDTA).

[0260] (xii) Pass cells through column and then wash with 4×500 μl withHBSS (containing 2% FCS and 2 mM EDTA).

[0261] (xiii) Elute and collect cells in a conical tube, then pellet andresuspended in IMDM as in Section A (vi).

[0262] D) FACSVantage Purified B Cells:

[0263] (i) Obtain mononuclear cells from peripheral blood samples ofB-CLL patients, as described in Section A, above.

[0264] (ii) Stain these cells with a combination of CD19-PE andCD20-FITC conjugated monoclonal antibodies to identify the B cells.

[0265] (iii) On the basis of CD19/CD20 fluorescence, sort approximately10⁷ cells using a Beckton Dickenson FACS Vantage and argon laseremitting at 488 nm.

[0266] (iv) Wash purified cells with Hanks balanced salt solutioncontaining 50% FCS and then allow to recover overnight at 37° C. in ahumidified incubator at 5% CO₂.

[0267] (v) Pellet and resuspend cells as described in Section A (vi)above and then treat with CR3/43 as described in Section B above.

[0268] Preparing Stem Cells in Whole Blood Cells

[0269] Treatment of cells with pure CR3/43 (Dako) monoclonal antibody inwhole blood:

[0270] (i) Select patients with WBC counts of 30-200×10⁹/L (ranging from73-95% B lymphocytes).

[0271] (ii) Collect blood by venipuncture into citrate, EDTA- orpreservative free heparin containing tubes.

[0272] (iii) Add CR3/43 Antibody directly to whole blood, at a finalconcentration of 0.08-0.16 μg/10⁶ cells (e.g. if WBC count was 50×10⁹/Lthen 50 μl of CR3/43 monoclonal antibody, mouse IgG concentration of 159μg/ml, should be added per ml of blood).

[0273] (iv) Mix blood thoroughly and leave overnight at room temperaturein an incubator.

[0274] (v) Analyse blood cells 0 hr, 2 hr, 6 hr and 24 hr after theaddition of mAbs.

[0275] Note: Due to the homotypic aggregation of B cells and theformation of adherent cells in the bottom of the test tube, induced bymAb CR3/43, thoroughly mix and sample cells using wide-bore pipette tipsor 21-G needle before analysis.

[0276] In order to obtain a uniform population of cells throughout theanalysis, divide blood sample into separate aliquots prior CR3/43treatment.

[0277] Preparation for Analysis of Stem Cells Produced by TreatingCultured B Cells with CR3/43 Monoclonal Antibody.

[0278] Stem cells produced using the methods of the invention can beassessed at a number of times points, for example every 2 hr, 7 hr, 24hr, daily, 7 days or longer periods (months, following weekly feeding ofcells with long term culture medium). In order to analyse all cells inthe well including adherent and non-adherent layer, together orseparately, one well has to be sacrificed.

[0279] (i) Gently remove non-adherent layer using a wide bore pipetteand disrupt cell clumps by repeated aspiration through 21-G needle toobtain single cell suspension.

[0280] (ii) Using a cell scraper scrape adherent layer and disruptgently cell clumps to obtain single cell suspension by repeatedaspiration through a 21-G needle.

[0281] (iii) Alternatively, trypsinize adherent layer by first rinsingwith HBSS and then adding 2 ml of 0.25% trypsin per well and incubate at37 C.° for 10 minutes.

[0282] (iv) Gently disturb cell clumps by repeated pipetting.

[0283] (v) After 10 mins incubate with 20% of FCS to a finalconcentration to inactivate the trypsin.

[0284] (vi) Wash cells twice with IMDM and 2% FCS by centrifugation at800 g for 10 mins.

[0285] (vii) Count cells and assess viability.

[0286] Analysis of Stem Cells:

[0287] The following methods can be used for the assessment of stemcells.

[0288] (A) Immunophenotype:

[0289] For Immunophenotypic analysis (using Flow Cytometry).

[0290] (i) For whole blood samples, immunostain (according tomanufacturer instructions), lyse the erythrocytes and wash the cellsafter the incubation period and treat with mAb. Lysing and washsolutions from Becton Dickinson may typically be used.

[0291] (ii) Leukocytes (in whole blood, mononuclear fraction, MACSmicrobeads negatively selected B cells or sorted B-CLL) should be eitherdoubly or singly labelled with mAbs conjugated directly to fluoresceinisothiocyanate (FITC) or phycoerythrin (PE).

[0292] (iii) Perform double labelling using IMK+ kit (Becton Dickinson):consisting of the following monoclonal antibody pairs:

[0293] CD45-FITC and CD14-PE;

[0294] CD19-PE and CD3-FITC;

[0295] CD8-PE and CD4-FITC;

[0296] HLA-DR-PE and CD3-FITC; and

[0297] CD56, CD16-PE and CD3-FITC.

[0298] Also isotype match negative controls for IgG₁-FITC andIgG_(2a)-PE are included.

[0299] (iv) The following additional antibodies can also be used whichare manufactured by Dako and Becton Dickinson:

[0300] PE-conjugated: anti-CD8, anti-CD33, anti-13, anti-CD34,anti-CD19, anti-CD2, anti-CD14, anti-CD33 and anti-CD5;

[0301] FITC-conjugated: anti-CD3, anti-CD7, anti-IgM, anti-CD22anti-CD20, anti-CD10, anti-CD7, anti-CD16, anti-TCRαβ.

[0302] (v) The following can also be used.

[0303] Also affinity purified IgG₃ mAb specific for CD34 (Dako) can beused and is detect with FITC- or PE-labelled goat anti-mouseimmunoglobulin F(ab)′₂ fragment as secondary antibody (DAKO).

[0304] Quantum Red (PE-Cy5)-conjugated anti-CD34 (Dako) was also used.

[0305] (vi) Analyse cells using FACScan or FACS Vantage (BectonDickinson).

[0306] (vii) Analyse data using Proprietary Paint-a-Gate, Lysis II,Consort 30 and CellQuest software.

[0307] (B) Morphology:

[0308] For morphological analysis:

[0309] Light Microscopy

[0310] (i) Resuspend cells thoroughly using wide-bore pipette tips or21-G needle.

[0311] (ii) Examine under a Leitz microscope using Wright's or Giemsastains.

[0312] (iii) Morphological analysis of B-CLL lymphocytes can beperformed in blood films or cytocentrifuged preparations, respectively.

[0313] Confocal Microscopy

[0314] (i) Obtain B cells as described above (B-CLL or healthy B cellsobtained from buffy coat of healthy blood donors)

[0315] (ii) Treat B cells with CR3/43 monoclonal antibody as describedabove.

[0316] (iii) Add 2 ml of cell suspension to an organ culture dish (Thebottom of this dish is engineered to have a cover-slip).

[0317] (iv) Add 15 μl of monoclonal antibody to CD19 FITC-conjugate and15 μl of monoclonal antibody to CD34 PE/Cy 5-conjugate (Quantum Red).

[0318] (v) Use Propidium Iodide to assess viability and Hoechst to stainthe nuclei.

[0319] (C) PCR Analysis of VDJ Gene Rearrangement

[0320] The VDJ region of the IgH gene was analyzed by PCR (Perkin elmerthermal cycler) using template DNA from B-CLL peripheral blood samplesbefore and after (2 hr, 6 hr and 24 hr) antibody treatment. The β-actingene was used as a control. For the VDJ region, V_(H)1, V_(H)2, V_(H)3,V_(H)4, V_(H)5 and V_(H) ⁶ family-specific sense primers were used withJ antisense primers (Deane and Norton, 1990). All primers weresynthesized by the Molecular Biology Unit, Randall Institute, King'sCollege, London.

[0321] (D) Southern Analysis of VDJ Gene Rearrangement

[0322] (i) Digest the Genomic DNA from treated and untreated peripheralblood samples or purified B cells (from B-CLL patients), usingBamHI/HindII—typically cells from a number of wells are required to givea sufficient amount of DNA to conduct the analysis.

[0323] (ii) The digests were resolved on 0.8% agarose gels andtransferred to GeneScreeng® nylon membranes (Dupont) according tomanufacturer's instructions (Southern, 1975).

[0324] (iii) The rearrangement of the IgH gene can be characterised byanalysing the J region of the IgH locus, using ³²P-labeled human J_(H)DNA probe isolated from placental genomic DNA (Calbiochem, OncogeneScience).

[0325] (iv) Autoradiographs should be kept at −70° C. for several daysprior to developing.

[0326] (E) Long Term Culture:

[0327] Cell cultures prepared as described above can be maintained forlonger periods (long term culture) by weekly feeding using long termculture medium (IMDM, 10% FCS, 10% HS, 1% hydrocortisone 5×10⁻⁷M stocksolution).

[0328] (i) First, following 24 hr from the initiation of CR3/43treatment dilute cells in each well by adding 2 mls of long term culturemedium.

[0329] (ii) Feed wells weekly following removal of half of the growthmedium.

[0330] (iii) Inspect wells using phase-contrast microscopy.

[0331] (F) Colony Forming Assays:

[0332] (i) After, 24 hr following initiation (or longer incubationperiod with pure monoclonal antibody CR3/43) of treatment, obtain 300 μlin culture medium of the non-adherent cells as described above.

[0333] (ii) Add to the cell suspension in the culture medium above, 3mls of methocult GFH4434 (StemCell Technologies, consisting ofmethylcellulose in Iscove's MDM, FCS, BSA, L-glutamine, rh stem cellfactor, rh GM-CSF, rh IL-3 and rh erythropoietin).

[0334] (iii) Take 1.1 ml of cell mixture and plate in triplicate.

[0335] (iv) Incubate the plates at 37° C. in a humidified petri dishwith 5% CO₂ and 5% O2 for 14 days.

[0336] (v) Inspect the wells before and after treatment with CR3/43monoclonal antibody using phase-contrast microscopy.

[0337] B. Results

[0338] CD19 and CD3 Panel

[0339] Treatment of blood samples with monoclonal antibody to thehomologous region of the β-chain of the HLA-DR antigen always decreasedthe relative number of CD19⁺ cells. This marker is a pan B-cell antigen(see Tables). This antigen is present on all human B lymphocytes at allstages of maturation but is lost on terminally differentiated plasmacells. Hence, this is an indication that B cells wereretrodifferentiating into undifferentiated cells.

[0340] The same treatment caused the relative number of CD3⁺ cells toincrease dramatically especially in blood of patients with B-CLL, whichwas always accompanied by an increase in the relative number inCD3⁻CD19⁻ cells. CD3 is present on all mature T-lymphocytes and on65%-85% of thymocytes. This marker is always found in association withα-/β- or gamma/delta T-cell receptors (TCR) and together these complexesare important in transducing signals to the cell interior. Hence, thisis an indication that B cells were retrodifferentiating intoundifferentiated cells and then being committed to new differentiatedcells, namely T cells.

[0341] A novel clone of cells appeared in treated blood of B-CLLpatients co-expressing the CD19 and CD3 markers—i.e. CD19⁺ and CD3⁺cells (see Chart 1, patient 2, 3 & 4 at 2 hr, 6 hr & 24 hr of startingtreatment). Other patients with different conditions showed an increasein the relative number of these clones of cells. These cells wereexceptionally large and heavily granulated and extremely high levels ofCD19 were expressed on their cell membrane. The CD3 marker seems to beexpressed on these cells at similar levels to those expressed on normalmature lymphocytes.

[0342] In Table 2, patient numbers 2, 3 and 4 are actually numbersrepresenting the same patient and their delineation was merely to showthe effect of treatment on blood with time (See Table 1 for experimentaland clinical condition of this patient).

[0343] The CD19⁺CD3⁺ clones in treated samples seem to decrease withtime, reaching original levels to those determined in untreated sampleat 2 hrs, 6 hrs and 24 hrs.

[0344] Another type of cell of the same size and granulity was detectedin treated samples and these cells had high levels of CD19 expressed ontheir surface but were negative for the CD3 marker and rich in FCreceptors. However, the relative number of these cells appeared todecrease in time. Of interest, at 24 hours treatment of blood sample (2,3 and 4) there was a decrease in the relative number of CD19⁻CD3⁻ cellsin a group of cells that were initially observed to increase after 2 and6 hrs treatment of blood samples. However, Coulter counts of WBCpopulations were reduced on treatment of blood with monoclonal antibodyto the homologous region of the β-chain of the HLA-DR antigen. Thisfinding suggests that this type of treatment gives rise to atypicalcells that cannot be detected by Coulter (Table 1) but can be accountedfor when measured by flow cytometry which counts cells on the basis ofsurface markers, size and granulity. Furthermore, these atypical cellswere accounted for by analysing morphology using Wright's stain under amicroscope. Flow cytometric charts of these phenomena are represented inCharts (1, 2, 3 & 4) and the immunophenotypic changes obtained ontreatment of blood samples seems to suggest that CD19⁺ and CD3⁺lymphocytes are an interconnected group of cells but remain distinct onthe basis of CD19 and CD3 relative expression compared to stem cells.

[0345] In Table 2, patient numbers 5 and 6 represent the same patientbut analysis of treated and untreated blood samples were monitored withtime and at the same time (see Table 1).

[0346] Patients' blood with no B-cell malignancy showed similar trendsof immunophenotypic changes when compared to blood of B-CLL patients butthe changes were not to the same extent. However, the relative andabsolute number of B-lymphocytes and MHC class II positive cells in theblood of these patients are extremely low compared to those found in theblood of B-CLL patients.

[0347] Two brothers both with X-linked infantile hypogammaglobulinemiawho were B cell deficient showed different immunophenotypic changes inthe relative number of CD3⁺ cells on treatment of their blood. Theyounger brother who was 2 months old and not ill, on treatment of hisblood, showed a slight increase in the relative number of CD3⁺ cellswhich was accompanied by a decrease in the relative number of CD3⁻CD19⁻cells. On the other hand, the other brother who was 2 years old and wasextremely sick and with a relatively high number of activated T cellsexpressing the DR antigens showed a decrease in the number of CD3⁺ cellson treatment of his blood. No other markers were used to measure otherimmunophentypic changes that might have occurred because the bloodsamples obtained from these two patients were extremely small (Table 2,ID-43/BD and 04/BD).

[0348] Patient 91 in Table 2 shows a decrease in the relative number ofCD3⁺ cells following treatment of blood which was accompanied by anincrease in the relative number of CD3⁻CD19⁻ cells. However, on analysisof other surface markers such as CD4 and CD8 (see Table 3) the patientwas observed to have a high relative number of CD4⁺CD8⁺ cells in hisblood and this was noted prior to treatment of blood samples withmonoclonal antibody to the β-chain of the DR antigen and these doublepositive cells decreased appreciably following treatment of blood.Furthermore, when further markers were analyzed the relative number ofCD3⁺ cells were seen to have elevated (See Table 4).

[0349] An enriched preparation of B-lymphocytes obtained from healthyblood donors when treated with monoclonal antibody to the β-chain of DRantigens showed a dramatic increase in the relative number of CD3⁺ cellswhich were always accompanied by a decrease in the relative number ofCD19⁺ cells and by an increase in the relative number of CD19⁻CD3⁻cells. Further analysis using markers such as CD4 and CD8 show aconcomitant increase in the relative number of these markers. However,an enriched preparation of T lymphocytes of the same blood donors whentreated with the same monoclonal antibody did not show the same changes.

[0350] CD4 and CD8 Panel

[0351] The CD4 antigen is the receptor for the human immunodificiencyvirus. The CD4 molecule binds MHC class II antigen in the B2 domain, aregion which is similar to the CD8 binding sites on class I antigens.Binding of CD4 to class II antigen enhances T cell responsiveness toantigens and so does the binding of CD8 to class I antigens. The CD8antigens are present on the human supressor/cytotoxic T-lymphocytessubset as well as on a subset of natural killer (NK) lymphocytes and amajority of normal thymocytes. The CD4 and CD8 antigens are coexpressedon thymocytes and these cells lose either markers as they mature intoT-lymphocytes.

[0352] On analysis of the CD4 and CD8 markers—see below—and from amajority of blood samples presented in Table 2, a pattern of stainingemerges which supports the presence of a retrodifferentiation process ofB-lymphocytes into undifferentiated cells and the subsequentdifferentiation into T-lymphocytes.

[0353] CD4⁺CD8⁺ cells, which are double positive cells, always appearedfollowing treatment of blood samples with monoclonal antibody to thehomologous region of the β-chain and these types of cells were markedlyincreased in the blood of treated samples of patients with B-CLL andwhich were absent altogether in untreated samples (See Table 3 andCharts 1, 2, 3 & 4). In the same specimens the relative number of singlepositive cells such as CD8⁺ and CD4⁺ cells was also noted to increasesimultaneously. Furthermore, a decrease in the relative number ofCD4⁻CD8⁻ cells which, at least in the case of B-CLL correspond to Bcells was noted to fall dramatically in treated samples when compared tountreated specimens which remained at the same level when measured withtime. However, measurement of the relative number of CD4⁺CD8⁺ cells withtime in treated samples showed that there was a concomitant increase inthe number of single positive cells with a decrease in the relativenumber of double positive cells. This type of immunophenotypic change ischaracteristic of thymic development of progenitor cells of theT-lymphocyte lineage in the thymus (Patient number 2,3 and 4). The CD4antigen is present on the helper/inducer T-lymphocyte subsets (CD4⁺CD3⁺)and a majority of normal thymocytes. However, this antigen is present inlow density on the cell surface of monocytes and in the cytoplasm ofmonocytes and macrophages (CD3⁻CD4⁺).

[0354] The relative number of CD4⁺ low cells was affected differently indifferent blood samples following treatment. The relative number of thistype of cells seems unaffected in blood samples of patients with B-CLLfollowing treatment when compared to untreated samples. Such low levelsof CD4 expression is found on monocytes and very early thymocytes.

[0355] Patient HIV⁺25 on treatment showed a substantial increase in thenumber of double positive cells expressing CD4 and CD8 simultaneously.On the other hand, patient 91 on treatment showed a decrease in thissubtype of cells and the observation of such phenomenon is timedependent. The relative number of CD8⁺ cells was observed to increase inuntreated blood samples of patients with B-CLL when measured with timewhereas the relative number of CD4⁺ and CD4⁺ low cells was observed todecrease at the same times (Table 3 patient 2, 3 and 4).

[0356] DR and CD3 Panel

[0357] The DR markers are present on monocytes, dendritic cells, B-cellsand activated T-lymphocytes.

[0358] Treated and untreated samples analysed with this panel showedsimilar immunophenotypic changes to those obtained when blood sampleswere analysed with the CD19 and CD3 markers (see Table 2) and theseantigens as mentioned earlier are pan B and T-cell markers respectively.

[0359] Treatment of blood with monoclonal antibodies seems to affect therelative number of DR⁺ B-lymphocytes so that the level of DR+ cellsdecrease. In contrast, the relative number of CD3⁺ (T-cells) cellsincrease significantly (see Table 4 and Chart). Furthermore, therelative number of activated T cells increased in the majority oftreated blood samples of patients with B-CLL and these types of cellswere affected variably in treated samples of patients with otherconditions. Furthermore, the relative number of DR high positive cellsappeared in significant numbers in treated samples of patients withB-CLL and a 6 day old baby with increased DR⁺CD34⁺ blasts in his blood.However, it should be noted that the blasts which were present in thispatients blood were negative for T and B-cell markers before and aftertreatment but became more positive for myeloid lineage antigensfollowing treatment. The relative number of CD3⁻DR⁻ cells increased inthe majority of treated blood samples and was proportional to increasesin the relative number of CD3⁺ cells (T-cells) and was inverselyproportional to decreases in the relative number of DR+ cells (B-cells).

[0360] CD56&16 and CD3 Panel

[0361] The CD56&CD16 markers are found on a heterogeneous group ofcells, a subset of lymphocytes known generally as large granularlymphocytes and natural killer (NK) lymphocytes. The CD16 antigen isexpressed on virtually all resting NK lymphocytes and is weaklyexpressed on some CD3⁺ T lymphocytes from certain individuals. Thisantigen is found on granulocytes in lower amount and is associated withlymphocytes containing large azurophilic granules. The CD16 antigen isthe lgG FC receptor III.

[0362] A variable number of CD16⁺ lymphocytes coexpress either the CD57antigen or low-density CD8 antigen or both. In most individuals, thereis virtually no overlap with other T-lymphocyte antigens such as theCD5, CD4, or CD3 antigens. The CD56 antigen is present on essentiallyall resting and activated CD16⁺ NK lymphocytes and these subsets ofcells carry out non-major histocompatibility complex restrictedcytotoxicity.

[0363] Immunophenotyping of treated and untreated blood samples of B-CLLand some other patients with other conditions showed an increase in therelative number of cells coexpressing the CD56&CD16 antigens which wereheavily granulated and of medium size (see Table 5 and Charts 1, 2, 3 &4). These observations were also accompanied by a marked increase in therelative number of cells expressing the CD3 antigen only (without theexpression of CD56 and CD16 markers) and cells coexpressing theCD56&CD16 and CD3 markers together.

[0364] In Table 5, patient numbers 2, 3, and 4 represent the same bloodsample but being analysed at 2 hours, 6 hours and 24 hours respectively(before and after treatment). This sample shows that treatment of bloodwith monoclonal antibody to the homologous region of the β-chain of DRantigen seems to cause spontaneous production of CD56⁺ and CD16⁺ cells,CD3⁺ cells and CD56⁺ and CD16⁺ CD3⁺ cells and these observations werealways accompanied by the disappearance of B-cell markers (CD19, DR,CD56, CD16⁻CD3⁻).

[0365] Onward analysis of this blood sample before and after treatmentshowed the levels of CD56⁺ and CD16⁺ cells to decrease with time and thelevel of CD3⁺ cells to increase with time.

[0366] Blood samples of patient 7 with B-CLL, did not show any changesin the number of cells expressing the CD56, CD16 and CD3 antigens whencompared to immunophenotypic changes observed in treated and untreatedsamples and this is because the amount of monoclonal antibody added wasextremely low relative to the number of B lymphocytes. However,treatment of this patient's blood sample on a separate occasion with anappropriate amount of monoclonal antibody showed significant increasesin the relative number of CD3⁺, CD56⁺ & CD16⁺ and CD56⁺ and CD16⁺ CD3⁺cells.

[0367] Blood samples of other patients with other conditions showedvariable changes in the level of these cells and this seems to bedependent on the number of B-lymphocytes present in blood beforetreatment, duration of treatment and probably the clinical condition ofpatients.

[0368] CD45 and CD14 Panel

[0369] The CD45 antigen is present on all human leukocytes, includinglymphocytes, monocytes, polymorphonuclear cells, eosinophils, andbasophils in peripheral blood, thymus, spleen, and tonsil, and leukocyteprogenitors in bone marrow.

[0370] The CD14 is present on 70% to 93% of normal peripheral bloodmonocytes, 77% to 90% of pleural or peritoneal fluid phagocytes. Thisantigen is weakly expressed on granulocytes and does not exist onunstimulated lymphocytes, mitogen-activated T lymphocytes, erythrocytes,or platelets.

[0371] The CD45 antigen represents a family of protein tyrosinephosphatases and this molecule interacts with external stimuli(antigens) and effects signal transduction via the Scr-family membersleading to the regulation of cell growth and differentiation.

[0372] Engagement of the β-chain of the DR antigens in treated bloodsamples especially those obtained from patients with B-CLL suggests thatsuch a treatment affects the level of CD45 antigens on B-lymphocytes.The overall immunophenotypic changes that took place on stimulation ofthe β-chain of the DR antigen seem to give rise to different types ofcells that can be segregated on the basis of the level of CD45 and CD14expression as well as morphology as determined by forward scatter andside scatter (size and granulity respectively) and these results arepresented in Table 6 and Charts (1, 2, 3 & 4). See also FIG. 5 whichdemonstrates the appearance of CD45⁻CD14⁻ cells after treatment with theCR3/43 antibody. These cells are not haempoietic cells.

[0373] On treatment the relative number of CD45 low cells (when comparedto untreated samples) increased significantly and so did the relativenumber of cells co-expressing the CD45 and CD14 antigens. This type ofimmunophenotypic changes coincided with a decrease in the relativenumber of CD45 high cells (compared to untreated samples). However, thislatter population of cells can be further divided on the basis ofmorphology and the degree of CD45 expression. One type was extremelylarge and had extremely high levels of CD45 antigen when compared to therest of cells present in the charts (see charts 1, 2, 3 and 4). Onanalysis of this panel following treatment with time (see Table 6patient 2, 3 and 4 and chart 1) the relative number of CD45⁺ cellsinitially fell drastically with time to give rise to CD45 low cells.However, analysis of blood 24 hours later showed the opposite situation.

[0374] Samples 5 and 7 reveal opposite immunophenotypic changes to thoseobtained with other samples obtained from other B-CLL patients and thisis because the samples were analysed at a much earlier incubation timewith the monoclonal antibody. In fact the sequential analysis of bloodsamples after treatment seems to suggest that the immunophenotypicchanges undertaken by B lymphocytes is time dependent because itrepresents a stage of development and the immunophenotypic changesmeasured at time X is not going to be the same at time X plus (its notfixed once induced). However, these types of changes must be occurringin a more stringent manner in the body otherwise immunopathology wouldensue. The effect of treatment of blood samples from other patients withno B-cell malignancy show variable changes in immunophenotypes of cellsand this because B-lymphocytes are present in lower amount. However,treatment of enriched fractions of B-lymphocytes obtained from healthyblood donors show similar immunophenotypic changes to those obtainedwith B-CLL with high B lymphocyte counts.

[0375] CD8 and CD3 Panel

[0376] The CD8 antigenic determinant interacts with class I MHCmolecules, resulting in increased adhesion between the CD8+ Tlymphocytes and the target cells. This type of interaction enhances theactivation of resting lymphocytes. The CD8 antigen is coupled to aprotein tyrosine kinase (p56ick) and in turn the CD8/p56ick complex mayplay a role in T-lymphocyte activation.

[0377] Treatment of blood samples obtained from patients with B-CLL withmonoclonal antibody to the B chain causes a significant increase in therelative number of CD3CD8 and CD3 (highly likely to be CD4CD3) positivecells thus indicating more clearly that double positive cells generatedinitially are undergoing development into mature T-lymphocytes. This isa process that can be measured directly by CD19 and by DR and indirectlyby CD8⁻CD3⁻ antigens. Serial assessment of treated blood samples of thesame patient with time seems to agree with a process which is identicalto thymocyte development (Table 7, patient 2, 3 and 4 and Chart 1).

[0378] The relative number of CD8⁺ cells increased with time in treatedand untreated samples but to a higher extent in untreated samples. Onthe other hand, the relative number of CD8⁺CD3⁺ cells decreased withtime in untreated samples. However, the relative number of CD3⁺ cellsincreased in treated blood samples when measured with time and thesetypes of cells highly correspond to CD4⁺CD3⁺ single positive cells; amaturer form of thymocytes. In addition, since these samples were alsoimmunophenotyped with other panels (mentioned above in Tables 3, 4, 5and 6) the overall changes extremely incriminate B cells in thegeneration of T lymphocyte progenitors and progenies.

[0379] Blood samples from a patient with B-CLL (number 2, 3 and 4 Tables1, 2, 3, 4, 5, 6, 7) in separate aliquots were treated with nothing, PEconjugated monoclonal antibody to the homologous region of the β-chainof DR antigen and unconjugated form of the same monoclonal antibody. Oncomparison of PE conjugated treatment clearly indicates no change in therelative number of CD3 positive cells and associated markers such as CD4which have been observed in significant levels when the same bloodsample was treated with unconjugated form of the antibody. However, anincrease in the number of CD45 positive cells with no DR antigen beingexpressed on their surface was noted when measured with time (see Table8). A finding that was similar to that noted in untreated samples whenimmunophenotyped with time (Table 6). Furthermore, the relative numberof cells expressing CD45 low decreased in time, a phenomenon which wasalso noted in the untreated samples (when measured with time) of thesame patient (see chart 1A).

[0380] C. Comparison of the Effect of other Monoclonal Antibodies withDifferent Specificity on T-Lymphophoiesis

[0381] CD19 and CD3 Panel

[0382] Treatment of blood samples with monoclonal antibody to thehomologous region of the α-chain of the DR antigen and the homologousregion of MHC Class I antigens decreased the number of CD3⁺ cells andincreased the number of CD19⁺ cells. Treatment of the same blood withmonoclonal antibody to the homologous region of the β-chain of the DRantigen decreased the number of CD19⁺ cells and increased the number ofCD3⁺ cells. Treatment with the latter monoclonal antibody withcyclophosphoamide revealed the same effect (Table 14 patient 5/6 withB-CLL at 2 hr treatment).

[0383] Onward analysis of CD19⁺ and CD3⁺ cells in the same samplesrevealed further increases in the relative number of CD3⁺ cells only inblood treated with monoclonal antibody to the homologous region of theβ-chain of DR antigen (Table 14 patient 5/6 at 24 hours followingtreatment). However, onward analysis (24 hours later patient 5/6 Table14) of blood samples treated with cyclophosphamide plus monoclonalantibody to the β-chain of DR antigen show reversal in the relativenumber of CD19⁺ and CD3⁺ cells when compared to that observed at 2 hourincubation time under exactly the same condition.

[0384] In general, treatment of blood samples of the same patient withmonoclonal antibody to the homologous region of the α chain of the DRantigen or monoclonal antibody to the homologous of the α-chain of theclass I antigen shows an increase in the relative number of CD19⁺ cells(pan B marker) when compared to untreated sample. The relative number ofCD19⁻CD3⁻ cells decreased slightly in blood samples treated withmonoclonal antibody to the α-chain of DR antigen or treated withmonoclonal antibody to class I antigens (see Table 14 & Charts 2, 3 &4). Treatment of blood samples of patient 09 with monoclonal antibody toclass I antigens increased the relative number of CD3⁺ cells anddecreased slightly the relative number of CD19⁺ and CD19⁻CD3⁻ cells.However, treatment of an enriched preparation of B-lymphocytes obtainedfrom healthy blood donors with monoclonal antibody to the β-chain orα-chain of DR antigen showed similar immunophenotypic changes to thoseobtained with patient with B-CLL.

[0385] Treatment of HIV⁺ and IgA deficient patients with monoclonalantibody to the β-chain of the DR antigen increased the relative numberof CD3⁺ cells and decreased the relative number of CD19⁺ cells. However,treatment of the same blood sample with monoclonal antibody to thehomologous region of class I antigen did not produce the same effect.Treatment of blood samples obtained from patients (34/BD and 04/BD) withB-cell deficiency showed variable immunophenotypic changes when treatedwith monoclonal antibodies to the β-chain of the DR antigen, class Iantigens and CD4 antigen.

[0386] CD4 and CD8 Panel

[0387] Blood samples analysed using the CD19 and CD3 panel (Table 14)were also immunophenotyped with the CD4 and CD8 panel (Table 15). Bothpanels seem to agree and confirm each other. Incubation for 2 hours ofblood samples of patients with B-CLL (Table 15, patients 5/6 and 10,Charts 2, 3 & 4) with monoclonal antibody to the homologous region ofthe β-chain of the DR antigen or with this monoclonal antibody pluscyclophosphoamide increased the relative number of CD8⁺ and CD4⁺ cellsand cells coexpressing both markers. On the other hand, treatment of thesame samples with monoclonal antibodies to the homologous region of theα-chain of the DR antigen or the homologous region of the α-chain ofclass I antigen did not produce the same effects.

[0388] Comparison of immunophenotypic trends obtained at 2 hours and 24hours incubation periods with monoclonal antibody to the β-chain of theDR antigen plus cyclophosphoamide revealed reverse changes in therelative number of CD4 and CD8 positive cells (Table 15, patient 5/6with B-CLL at 2 hours and 24 hours) and such changes were in accordancewith those obtained when the same blood sample was analysed with theCD19 and CD3 panel (Table 14 the same patient). The later findingsindicate that the subsequent differentiation is reversible as theundifferentiated cells can differentiate into T-lymphocytes orB-lymphocytes.

[0389] DR and CD3 Panel

[0390] The immunophenotypic changes obtained with DR and CD3 (Table 16)panel confirm the findings obtained with CD19 and CD3 panel and CD4 andCD8 panel (Tables 14 & 15 & Charts 2, 3 & 4) which followed treatment ofthe same blood samples with monoclonal antibodies to the homologousregion of the beta- or alpha- side of the DR antigen or monoclonalantibody to class I antigens or monoclonal antibody to the β-chain ofthe DR antigen plus cyclophosphoamide at 2 hour analysis.

[0391] From the results, it would appear that the monoclonal antibody tothe homologous region of the β-chain of the DR antigen is extremelycapable of driving the production of CD3 positive cells from DR⁺ cells.

[0392] Furthermore, treatments such as those involving engagement of theα-chain of DR antigens or engagement of the β-side of the molecule inconjunction with cyclophosphoamide (prolonged incubation time) promotedincreases in the relative number of CD19⁺ cells or DR⁺ cells.

[0393] CD56&16 and CD3 Panel

[0394] Treatment of blood samples, especially of those of patients withB-CLL with high B-lymphocyte counts with monoclonal antibody to thehomologous region of the β-chain of the DR antigen increased therelative number of CD56&16 positive cells.

[0395] In these patients the relative number of CD3⁺ and CD56⁺ andCD16⁺CD3⁺ cells also increased following treatment of blood samples withmonoclonal antibody to the β-chain, confirming earlier observationsnoted with the same treatment when the same blood samples were analysedwith CD3 and CD19 and DR and CD3 panels.

[0396] CD45 and CD14 Panel

[0397] Blood samples treated with monoclonal antibodies to the β- oralpha- chains of the DR antigen or to the β-chain plus cyclophosphoamideor class I antigens were also analysed with the CD45 and CD14 panel(Table 18). The delineation of CD45 low, CD45 high and CD45 medium isarbitrary. Treatment of blood sample 5/6 (at 2 hours) with monoclonalantibodies to the β-chain of the DR antigen or with this monoclonalantibody plus cyclophosphoamide generated CD45⁺ low cells and increasedthe relative number of CD45⁺ medium cells. However, the former treatmentincreased the relative number of CD45⁺ high cells and the lattertreatment decreased the relative number of CD45⁺ medium cells and thesechanges appeared to be time dependent.

[0398] Blood samples of patient 5/6 and 10 (B-CLL) on treatment withmonoclonal antibody to class I antigens showed a decrease in therelative number of CD45⁺ medium cells and similar observations werenoted in blood samples 09 and HIV⁺ following the same treatment whencompared to untreated samples. Treatment of blood samples of HIV+ andIgA/D patients with monoclonal antibody to class I antigen increased therelative number of CD45⁺ low cells when compared to untreated samples orsamples treated with monoclonal antibody to the β-chain of the DRantigen. However, blood samples of these patients showed a decrease inthe relative number of CD45⁺ medium cells on treatment with monoclonalantibody to the homologous regions of the β-chain of the DR antigen.Medium CD45⁺ cells increased in blood samples of IgA/D patient followingmonoclonal antibody to class I antigen treatment. Cells that wereextremely large, heavily granular and expressing intense levels of CD45antigen were noted in treated blood samples with monoclonal antibody tothe homologous region of the β-chain of DR antigen of MHC class IIantigens (see Charts 1, 2, 3 & 4).

[0399] CD8 and CD28 Panel

[0400] The CD28 antigen is present on approximately 60% to 80% ofperipheral blood T (CD3⁺) lymphocytes, 50% of CD8⁺ T lymphocytes and 5%of immature CD3-thymocytes. During thymocyte maturation, CD28 antigenexpression increases from low density on most CD4⁺CD8⁺ immaturethymocytes to a higher density on virtually all mature CD3⁺, CD4⁺ orCD8⁺ thymocytes. Cell activation further augments CD28 antigen density.Expression of the CD28 also divides the CD8⁺ lymphocytes into twofunctional groups. CD8⁺CD28⁺ lymphocytes mediate alloantigen-specificcytotoxicity, that is major histocompatibility complex (MHC) classI-restricted. Suppression of cell proliferation is mediated by theCD8⁺CD28⁻ subset. The CD28 antigen is a cell adhesion molecule andfunctions as a ligand for the B7/BB-1 antigen which is present onactivated B lymphocytes.

[0401] Treatment of blood samples of patients (Table 19, patients 5/6and 8) with B-CLL with monoclonal antibody to the homologous region ofβ-chain of the DR antigen increased the relative number of CD8⁺, CD28⁺and CD8⁺CD28⁺ cells and all other types of treatments did not.

[0402] CD34 and CD2 Panel

[0403] The CD34 antigen is present on immature haematopoietic precursorcells and all haematopoietic colony-forming cells in bone marrow,including unipotent (CFU-GM, BFU-E) and pluripotent progenitors(CFU-GEMM, CFU-Mix and CFU-blast). The CD34 is also expressed on stromalcell precursors. Terminal deoxynucleotidyl transferase (TdT)⁺ B- andT-lymphoid precursors in normal bone are CD34⁺, The CD34 antigen ispresent on early myeloid cells that express the CD33 antigen but lackthe CD14 and CD15 antigens and on early erythroid cells that express theCD71 antigen and dimly express the CD45 antigen. The CD34 antigen isalso found on capillary endothelial cells and approximately 1% of humanthymocytes. Normal peripheral blood lymphocytes, monocytes, granulocytesand platelets do not express the CD34 antigen. CD34 antigen density ishighest on early haematopoietic progenitor cells and decreases as thecells mature. The antigen is absent on fully differentiatedhaematopoietic cells.

[0404] Uncommitted CD34⁺ progenitor cells are CD38⁻, DR⁻ and lacklineage-specific antigens, such as CD71, CD33, CD10, and CD5, whileCD34+ cells that are lineage-committed express the CD38 antigen in highdensity.

[0405] Most CD34⁺ cells reciprocally express either the CD45RO or CD45RAantigens. Approximately 60% of acute B-lymphoid leukaemia's and acutemyeloid leukaemia express the CD34 antigen. The antigen is not expressedon chronic lymphoid leukaemia (B or T lineage) or lymphomas. The CD2antigen is present on T lymphocytes and a subset of natural killerlymphocytes (NK).

[0406] The results are shown in Charts 2, 3 and 4.

[0407] Analysis of blood samples of a patient with B-CLL (Table 20,patient 5/6 at 2 hours) after treatment with monoclonal antibodies tothe β-chain of the DR antigen or the α-chain of the same antigenrevealed marked increases in the relative number of CD34⁺ and CD34⁺CD2⁺cells after treatment with the former antibody. Since the same bloodsamples were immunophenotyped with the above mentioned panels (seeTables 14 to 19 ) for other markers the increase in the relative numberof CD34⁺ and CD34⁺CD2⁺ cells observed here seems to coincide withincreases in the relative number of CD4⁺CD8⁺, CD8⁺CD3⁺ and CD4⁺CD3⁺single positive (SP) cells. Furthermore, these findings which seemexclusive to engagement of the β-chain of the HLA-DR antigen, are indirect support that the process is giving rise to T-lymphopoiesis via Blymphocyte regression.

[0408] On analysing the same treatment 24 hours later the CD34⁺ cellsseemed to decrease in levels to give rise to further increase in therelative number of T lymphocytes. The process of retrodifferentiationthat initially gave rise to T-lymphopoiesis can be reversed to give riseto B-lymphopoiesis. The former phenomenon was observed at 2 hoursincubation time with monoclonal antibody to the β-chain of the HLA-DRantigen plus cylophosphoamide, whereas the latter process was noted at24 hours incubation time with the same treatment in the same sample(Chart 2).

[0409] Treatment of blood samples of HIV⁺ patient (Table 20 patientHIV+) with monoclonal antibody to the β-chain of the HLA-DR antigenmarkedly increased the relative number of CD34⁺ and CD2⁺CD34⁺ cells andso did treatment of the same blood sample with monoclonal antibody tothe β-chain of the HLA-DR antigen and monoclonal antibody to the α-chainof the same antigen when added together. However, treatment of thisblood sample with monoclonal antibody to the α-chain of the HLA-DRantigen did not affect the level of CD34⁺ cells. Treatment of bloodsamples obtained from a 6-day old baby (BB/ST Table 20) who wasinvestigated at that time for leukaemia and who had very high number ofatypical cells (blasts) in his blood with monoclonal antibody to theβ-chain of the HLA-DR antigen, or monoclonal antibody to the α-chain ofthe same antigen or both monoclonal antibodies added together resultedin the following immunophenotypic changes.

[0410] On analysis of untreated blood samples the relative number ofCD34⁺ and DR⁺ cells were markedly increased and on treatment withmonoclonal antibody to the β-chain the relative number of CD34⁺ cellsfurther increased but were noted to decrease on treatment withmonoclonal antibody to the α-chain of the HLA-DR antigen or treatmentwith monoclonal antibodies to the α and β-chains of the molecule whenadded together. However, the latter treatment increased the relativenumber of CD34⁺CD2⁺ cells and the opposite occurred when the same bloodsample was treated with monoclonal antibody to the β-chain of the HLA-DRantigen alone. On analysis of treated and untreated blood aliquots ofthe same patient 24 hours later the relative number of CD34+ decreasedwith all above mentioned treatments except it was maintained at a muchhigher level with monoclonal antibody to the β-chain of the HLA-DRantigen treatment. The latter treatment continued to decrease therelative number of CD34⁺CD2⁺ cells 24 hours later.

[0411] These results indicate that engagement of the HLA-DR antigen viathe β-chain promotes the production of more CD34⁺ cells from CD2⁺CD34⁻pool or from more mature types of cells such as B-lymphocytes ofpatients with B-CLL and these results indicate that this type oftreatment promotes retrodifferentiation. However, immunophenotyping ofblood samples 24 hours later suggests that these types of cells seem toexist in another lineage altogether and in this case cells seem to existor rather commit themselves to the myeloid lineage which was observed onanalysis of treated blood sample with the CD7 and CD13&33 panel.

[0412] Morphology changes immunophenotypic characteristics ofB-lymphocytes of B-CLL and enriched fractions of healthy individuals(using CD19 beads) on treatment with monoclonal antibodies to homologousregions of the β-chain of MHC class II antigens. These were accompaniedby a change in the morphology of B-lymphocytes. B-lymphocytes wereobserved colonising glass slides in untreated blood smears weresubstituted by granulocytes, monocytes, large numbers of primitivelooking cells and nucleated red blood cells. No mitotic figures orsignificant cell death were observed in treated or untreated bloodsmears. The results of Table 20 also demonstrate a further importantfinding in that according to the method of the present invention it ispossible to prepare an undifferentiated cell by the retrodifferentiationof a more mature undifferentiated cell.

[0413] D. Microscope Pictures

[0414] In addition to the antigen testing as mentioned above, the methodof the present invention was followed visually using a microscope.

[0415] In this regard, FIG. 6 is a microscope picture of differentiatedB cells before the method of the present invention. FIG. 7 is amicroscope picture of undifferentiated cells formed by theretrodifferentiation of the B cells in accordance with the presentinvention wherein the agent was a monoclonal antibody to the homologousregions of the β-chain of HLA-DR antigen. The undifferentiated cells arethe dark stained clumps of cells. FIG. 8 is a microscope picture of thesame undifferentiated cells but at a lower magnification.

[0416] FIGS. 6 to 8 therefore visually demonstrate theretrodifferentiation of B cells to undifferentiated stem cells by themethod of the present invention.

[0417]FIG. 9 is a microscope picture of differentiated B cells beforethe method of the present invention. FIG. 10 is a microscope picture ofundifferentiated cells formed by the retrodifferentiation of the B cellsin accordance with the present invention wherein the agent used was amonoclonal antibody to the homologous regions of the β-chain of HLA-DRantigen. Again, the undifferentiated cells are the dark stained clumpsof cells. FIG. 11 is a microscope picture of the formation ofdifferentiated granulocyte cells from the same undifferentiated cells ofFIG. 10.

[0418] FIGS. 9 to 11 therefore visually demonstrate theretrodifferentiation of B cells to undifferentiated stem cells by themethod of the present invention followed by commitment of theundifferentiated cells to new differentiated cells being of a differentlineage as the original differentiated cells.

[0419] These microscopy experiments have also been performed with bloodfrom BCLL patients, treated with the CR3/43 monoclonal antibody asdescribed above. As discussed above, blood from BCLL cells is a usefulaid in studying the retrodifferentiation process because the bloodcontains higher than normal numbers of B lymphocytes. The results areshown in detail in FIGS. 12 to 15.

[0420]FIG. 12 shows at two different magnifications, an untreated bloodsample from a BCLL patient. The untreated B lymphocytes (blue cells)show typical morphology, i.e. condensed chromatin structure and sparsecytoplasm. The remaining cells are erythrocytes (red blood cells).

[0421] Treatment of blood samples with antibody CR3/43 leads initiallyto clustering of B lymphocytes into aggregates (FIG. 13).

[0422] The clustered B cells gradually lose their typical morphology,characterised by the formation of cobblestone-like-cell areas,decondensation of chromatin structure, appearance of prominent nucleoli,enlargement of cell volume and cytoplasmic basophilia typical ofundifferentiated cells (FIG. 14). Relaxed (decondensed) chromatinstructure is an important feature of undifferentiated cells as comparedto differentiated cells. This is likely to be due to a need for moreextensive access to transcriptional units to determine changes in geneexpression required for commitment along a given cell lineage. Bycontrast, it is well known that more differentiated cell have a morecondensed chromatin structure since only a small amount of chromatinneeds to be transcriptionally active.

[0423] The appearance of undifferentiated cells is always accompanied bythe appearance of cells (15A to 15J) with differentiated morphology.Importantly, these cells could not have arisen by proliferation, since(i) the incubation time was too short for one or more complete celldivisions to take place (ii) no mitotic figures are seen and (iii) theabsolute number of leucocytes remained the same before and aftertreatment. Furthermore less differentiated progenitors were seen inassociation with their more differentiated progenies (see the myeloidprecursor in FIG. 15J), indicating that these specialized cells arose bydifferentiation.

[0424] Micrographs FIGS. 15A to 15J show the types of differentiatedcells seen following treatment of B-CLL lymphocytes with CR3/43monoclonal antibodies: Platelets (Pl)—FIG. 15A, Neutrophils (Ne)—FIG.15B, Eosinophils (Eso)—FIG. 15C, Megakaryocytes (Meg)—FIG. 15D,Basophils (Ba)—FIG. 15G, Lymphocytes (Ly)—FIG. 15H, Monocytes (Mo)—FIG.15I and Myeloid progenitors (Mp)—FIG. 15J. Also seen were erythroidprogenitors and macrophages (data not shown).

[0425] Thus, in summary, these microscopy results show changes in B cellmorphology in samples from BCLL patients, who have high levels of matureB lymphocytes. The microscopy pictures show changes in the morphology ofthe B lymphocytes, which initially cluster, followed by the appearanceof various cells with a graded range of morphologies from progenitorcells to differentiated cells (neutrophils, basophils, eosinophils,megakaryocytes, platelets, lymphocytes, macrophages, granulocytes, stabgranulocytes and stromal-like cells).

[0426] In addition, and very importantly, the presence of erythroid andmyeloid progenitors is seen (FIG. 15J—and data not shown). The myeloidprogenitor is clearly distinguishable morphologically from the othercells, being larger and with a distinct nuclear morphology as well ascontaining cytoplasmic granules.

[0427] The microscopy data therefore support morphologically what theflow cytometry data indicate in terms of cell surface markers. Thesedata allow one to conclude that treatment of B lymphocytes with anantibody to MHC HLA-DR β chain results in a decrease in the numbers of Blymphocytes and an increase in the number of cells of other haemapoieticlineages including immature precursor cells.

[0428] The retrodifferentiation of T cells treated with an antibody toan MHC class II α-chain (monoclonal antibody TAL.1B5) toundifferentiated stem cells by the method of the present inventionfollowed by commitment of the undifferentiated cells to newdifferentiated cells being of a different lineage as the originaldifferentiated cells was also followed by microscopy (data not shown).

[0429] E. Analysis of VDJ Recombination Rearrangements inRetrodifferentiated Lymphocytes

[0430] By way of background, the differentiated cells used in theseexperiments (B lymphocytes or cells with certain properties of Tlymphocytes) have genes which have already undergone rearrangement toencode a mature Ig or a TCR, respectively. In the process orrearranging, intermediate portions of DNA that are not part of thefinal, expressed TCR or Ig gene, primarily DNA which is between thevariable (V) region encoding segment and the constant (C)region-encoding segment of these receptors, are spliced out of thegenome. These excised fragments are retained in the cell in the form ofextrachromosomal DNA. For the cells to truly retrodifferentiate, theexcised DNA would be reinserted into the genome, placing the cells in astate similar to that preceding their original differentiation. Becauseof this, a probe complementary to a sequence in the rearranged gene willbe expected to hybridize to a larger DNA restriction fragment when theDNA has returned to its unrearranged or germ line state as compared tothe rearranged DNA that characterizes the differentiated state.

[0431] 1. Rearrangement of TCR Genes in Daudi Cells

[0432] In the experiment resulting in the Southern blot shown in FIG.16, a well-known cell line, Daudi, a B-cell lymphoma with one rearrangedTCR gene (and the other deleted), was used. Genomic DNA was preparedfrom Daudi cells and digested with EcoRI, subjected to gelelectrophoresis and probed with a labeled TCR β-chain DNA probe. Daudicells were used rather than B lymphocytes purified from human patientsbecause these cells are clonally related and form a homogenous cellpopulation with the same gene rearrangements that can be clearly viewedby Southern blotting of digested genomic DNA. In a normal blood sample,different cells have different rearrangements and so a Southern blotwould appear as a smear.

[0433] A functional gene encoding the TCR β-chain is assembled inlymphocytes by a series of somatic rearrangements that occur duringlymphocyte maturation to bring together a V segment, a D segment and a Jsegment. A very clear explanation of these rearrangement processes isgiven in Genes VI, Lewin, Oxford University Press, 1997 (pages1994-1023)—a standard undergraduate textbook. Particular pages are citedbelow.

[0434] Firstly, a D segment is joined by a recombination process to oneof several J segments in a D-J joining reaction. Then, one of the manypossible V segments(<60) is joined to the resulting DJ segment (V-Djoining) to form a complete TCR β-chain gene. The constant region geneis immediately downstream of the rearranged VDJ segment, although theremay be intervening J segments which are spliced out during RNAprocessing to bring the constant gene exon into proximity with therearranged VDJ gene segment (Lewin, p998).

[0435] In human cells, there are two different TCR β-chain constantregion gene segments, denoted Cβ1 and Cβ2, present at two differentloci, each of which is preceded by a cluster of six or seven joiningregion (Jβ) gene segments (Jβ1 and Jβ2) and one D segment (Dβ1 and Dβ2)(see FIG. 1, Toyonaga et al., 1985, Proc. Natl. Acad. Sci. USA 82:8624-8628 and Lewin, p1017).

[0436] The recombination events which lead to the V, D and J-C segmentsbeing brought into proximity are catalysed by a multitude of proteins,including RAG-1 and RAG-2 which recognise nonamer and heptamer sequencespresent at the recombining ends of the V, D and J-C gene sequences.Depending on the orientation of these nonamer/heptamer sequences,recombination results either in an inversion or a deletion. Both typesof events will result in a change in the restriction enzyme fragmentpattern of the genomic DNA. Furthermore, a deletion event does notnecessarily result in complete loss of the excised fragment. Rather, theends of the excised fragment are rejoined to produce a circle of DNAwhich remains in the cell (Okazaki et al., 1987, Cell 49: 477-85; Daviset al., 1991, J. Exp. Med. 173: 743-6; Livak and Schatz, 1996, Mol. CellBiol. 16: 609-18; Harriman et al. 1993, Annu Rev Immunol. 11:361-84).Each gene segment, of course, has two alleles since cells have a diploidchromosome complement.

[0437] In the normal germline state, the Cβ1 and Cβ2 genes are arrangedas shown in FIG. 1, Toyonaga et al., 1985. A restriction digest ofgenomic DNA with EcoRI will generate two relevant bands detectable bythe probe used in the experiment (the probe is a labelled DNA fragmentderived from Cβ1 which also hybridises to Cβ2 due to a high degree ofsequence homology): (i) a 12 kb band containing Cβ1 sequence; and (ii) a4 kb band containing Cβ2 sequence. This germline configuration is seenin undifferentiated immature cells (lane A of FIG. 16) This germlineconfiguration is also perfectly illustrated by lane 3 (2 hours withCR3/43 antibody) of FIG. 16, giving an identical pattern to that of laneA.

[0438] In the differentiated state, both alleles of Cβ1 and Cβ2 genesare rearranged such that there is no longer a 12 kb fragment at the Cβ1locus or a 4 kb fragment at the Cβ2 locus. In fact, no hybridisingfragment derived from the Cβ1 locus is present on the gel (this is dueto deletion of the hybridising sequence from both Cβ1 alleles as aresult of recombination). As for the Cβ2 locus, there are actually nowtwo major bands corresponding to different “alleles” resulting fromrearrangements on both chromosomes. The largest band, which is smallerthan 4 kb, corresponds to a fragment of one of the two rearrangedalleles. The lowest band is a fragment of the other rearranged allele.The intermediate minor band is probably derived from a subclone of Daudicells with a different rearrangement—hence its presence in a submolaramount to either allele. Nonetheless, the rearranged state is veryclearly shown in lane 1 where both major bands are clearly visible.

[0439] 2 hours with the negative control antibody (TAL.1B5) which bindsto the α-chain of MHC-DR actually results in the loss of the upper band,whereas the lowest band has a similar intensity to the untreated cellsin lane 1 (see lane 2). A possible explanation for this is that thecells are differentiating, further resulting in a further recombinationevent at the Cβ2 locus of one allele, which leads to loss of Cβ2sequences. This is entirely consistent with known phenomena.

[0440] 24 hours with the negative control antibody appears to restorethe three bands seen in the untreated cells (see lane 4). However thebands actually migrate at a lower position than the bands seen in lane2. It is not quite clear how this has arisen. A possible explanation isthat reintegration of deleted sequences has occurred, consistent withthe looping-out-excision-reintegration model (Malissen et al., 1986,Nature 319: 28-32). Nonetheless, neither result seen with the TAL.1B5antibody at 2 hours or 24 hours is indicative of a rearrangement to thegermline pattern. Lanes 2 and 4 actually represent a negativecontrol—the antibody to the α-chain does not result in restoration ofthe germline sequences.

[0441] By contrast, the results obtained with a monoclonal antibody(CR3/43) to the β-chain of MHC-DR after two hours show a pattern ofbands that correspond to the germline configuration, namely a 12 kb bandand a 4 kb band (compare lane 3 with lane A). In other words, theseresults show that the germline restriction pattern at the Cβ1 and Cβ2loci has been restored for all alleles.

[0442] From these results we conclude that the pattern of bands seen inlane 3 are indicative of a rearrangement of the genomic DNA of thedifferentiated cells to regenerate the germline configuration.

[0443] The importance of this finding should not be understated. Neverbefore has it been demonstrated that a genomic rearrangement, includingdeletions, can be reversed to restore the genome to the state in whichit existed before the differentiation process took place. The mostlikely explanation is that the inversion caused by the rearrangement ofthe Cβ2 alleles during differentiation has been reversed, and thedeletion of the Cβ1 sequence that caused loss of the 12 kb bands hasalso been reversed. The source of the missing Cβ1 sequence is likely tobe episomal circular DNA present in the nucleus from the originaldeletion event. The existence of this circular DNA has been cataloguedin the prior art (see references cited above). Nonetheless, the precisemechanism by which this restoration of the germline genome has occurredis not important. What is important is that it has occurred.

[0444] A continued incubation with the monoclonal antibody (CR3/43) tothe β-chain of MHC-DR for 24 hours results in a more complex bandingpattern (lane 5). However these bands do not represent the same bands asin the untreated control. In particular, fragments of about 12 kb thathybridize to the probe are still present (“Cβ2 alleles”). Further, it isimportant to appreciate that the bands marked “Cβ2 alleles” do notcorrespond to the smaller than 4 kb band seen in the untreated control(lane 1). The most likely explanation for the results seen in lane 5 isthat a secondary rearrangement process has occurred since thehybridization pattern resembles that of T-cells in that it ischaracterized by a rearranged TCR gene (this explanation is consistentwith the flow cytometry data showing an increase in cells having cellmarkers characteristic of T cells). Nonetheless, regardless of theprecise molecular explanation, the results seen in lane 5 at 24 hoursexposure to the CR3/43 antibody are supportive of the results obtainedat 2 hours exposure in lane 3.

[0445] 2. Rearrangement of Ig Gene in B-CLL Cells

[0446] The Southern blot shown in FIG. 17B was obtained using peripheralblood cells from patients with chronic lymphocytic leukemia (B-CLL).Genomic DNA was prepared from these largely monoclonal B cells anddigested with BamHI and HindIII, subjected to gel electrophoresis andprobed with a labeled TCR DNA probe. These B-CLL cells were treated for24 hours with the CR3/43 (anti class II MHC chain of HLA-DR, DP and DQ)which was described above. The blots were probed with a radiolabeled IgJ region probe. The two bands obtained from the untreated cells in laneA, represent the two rearranged Ig alleles (paternal and maternal).These bands did not appear in lane B which shows the pattern 24 hoursafter antibody treatment of cells. In their place appeared a 5.4 kb bandcharacteristic of the germ line Ig gene.

[0447] In another experiment, shown in FIG. 17A, cells were leftuntreated or treated for the times indicated with the anti-class II MHCβ-chain antibody. The Ig VDJ region was amplified by PCR in thedifferentiated (control) and antibody-treated B-CLL cells (left half ofgel). This generated a VDJ amplification product from the untreatedcells. However, no such band was observed in the antibody-treated cellsbecause, as a result of insertion of the excised genomic DNA, this “germline” DNA configuration was not susceptible to PCR amplification usingthe particular primers for VDJ. A similar experiment (right side of gel)allowed me to visualize the behavior of a control, housekeeping, geneencoding β-actin. There was no difference in the β-actin PCRamplification product, regardless of treatment. Thus, this “control”gene did not appear to be affected by the retrodifferentiation processthat caused profound alterations in the Ig gene of the same cells underthe same conditions.

[0448] The results presented above show that treatment of cells with anagent that engages an appropriate cell surface receptor inducesretrodifferentiation of these cells that is proven at the molecularlevel (and monitored) by observing the retrogression of therearrangements of chromosomal DNA that characterize the differentiatedstate. Thus, it is concluded on the basis of the molecular genetic andmorphological evidence that cells of the B lymphocyte lineage, treatedwith an agent (mAb) that engages the class II MHC β-chain, undergoretrodifferentiation. By contrast, the same cells treated withantibodies that engage class II α-chain are not similarly induced toretrodifferentiate. If anything, they appear to differentiate (forward)along the B cell pathway.

[0449] F. Further Studies on Retrodifferentiation of B Lymphocytes

[0450] FACsVantage puried BCLL cells (95% pure B cells0 from BCLLpatients were treated with the CR3/43 antibody as described above andthe cells processed by flow cytometry. The results shown below in TableA confirm further the results obtained above. A significant increase inthe number of CD34⁺ cells was obtained together with a large reductionin the number of cells having cell surface markers characteristic of theB lymphocyte lineage (CD19, CD20 and CD22). An important point to notefrom Table A is that it also shows an increase in the number of cellsthat are both CD34 negative and lineage negative. These undifferentiatedcells are not committed to the haemopoietic lineage and precede CD34⁺stem cells in differentiation. Further, examination of samples by lightmicroscopy showed a range of adherent cell types having morphologicalcharacteristics of non-haemopoietic cells. TABLE A Marker 0 hr 2 hr 24hr CD20 73 67 16 CD14 0 3 23 CD34 0 1 23 CD7 0 2 0 CD16 8 3 2 CD19 95 711 CD22 5 3 2 CD33 0 0 0 CD3 0 0 0

[0451] The loss of CD19 cell surface markers accompanied by theappearing of CD34 cell surface markers on the same cell has also beendemonstrated and recorded on video in real time using confocalmicroscopy. B-lymphocytes before the addition of CR3/43 mab stainedgreen with a FITC conjugated monoclonal antibody to CD19. After theaddition of CR3/43 mab, cells lost their green fluorescence and began tostain red with a PE/Cy5 (or quantum red) conjugated monoclonal antibodyto CD34 but not green (see FIG. 21 which shows two still images from thetimelapse video). The results clearly confirm that during B lymphocyteretrodifferentiation, lineage specific markers such as CD19 are lostwhilst a stem cell marker such as CD34 is re-expressed.

[0452] G. Other Agents that Induce Retrodifferentiation of B Lymphocytesto Haemopoietic Stem Cells

[0453] Initial studies actually identified threeagents—granulocyte/monocyte-colony stimulating factor (GM-CSF),erythropoietin and mAb CR3/43. A preparation of enriched, purified,normal B lymphocytes was treated with one of these three agents in asimilar manner to that described for CR3/43 and TAL.1B5 above andtreated samples examined by flow cytometry as described above. Comparedwith the negative control, all three samples treated with either GM-CSF,erythropoietin or mAb CR3/43 showed changes consistent withretrodifferentiation. In particular, all three agents increased therelative number of CD34⁺ cells in the cell population (see FIG. 18). Thegreatest effect, however, was seen with CR3/43 and consequently, thisagent was selected for use in the more detailed studies presentedherein.

[0454] H. Properties of Haemopoietic Stem Cells Produced by theRetrodifferentiation Process

[0455] Colony Forming Assays

[0456] To confirm that the CD34⁺ cells observed by flow cytometry andthe undifferentiated cells identified by microscopy had the propertiesof undifferentiated haemopoietic cells, blood samples treated with anantibody to the class II MHC β-chain (CR3/43—see above) were subjectedto colony forming assays—a standard method known in the art forassessing the capabilities of primitive haemopoietic cells.

[0457] In vitro clonal assays for hematopoietic stem cell allows thequantification of primitive progenitor cells that possess the ability toproliferate, differentiate and develop into phenotypically andfunctionally mature myeloid and/or erythroid cells. For example in thepresence of growth factors stem cell when seeded/immobilised in soft-gelmatrix in vitro are capable of clonal growth (proliferation) anddifferentiation.

[0458]FIG. 19 is a colony assay of stem cells produced according to themethods of the invention, using inverted bright-field microscopy. Inthis assay B cells obtained from buffy coat of healthy blood donors weretreated with CR3/43 mab and then subjected to colony assays as describedin the materials and methods section.

[0459] Panels (a) to (e) in FIG. 19 show:

[0460] a) Bright field microscopy of culture dish viewed at ×3magnification showing erythroid, myeloid and mixed (consisting of maturemyeloid and erythroid cells) colonies which can be seen readily even bythe naked eye. Each colony arose from a single haematopoietic stem cellby proliferation and subsequent differentiation.

[0461] b) MIX-CFC this colony arose from a single multi-potenthaematopoietic stem cell (stem cells capable of giving rise to cells ofmyeloid and erythroid lineages.

[0462] c) M-CFC this colony consists of macrophages.

[0463] d) GM-CFC this colony consist of the myeloid lineage includingmacrophages, granulocyte and megakaryocytes

[0464] e) BFU-E this colony consists of cells belonging to the erythroidlineage such as normoblasts and non-nucleated red cells. The redcolouration of cells shows that they are well hemogloblinized. The largesize of this colony indicates that it arose from an extremely primitivestem cell.

[0465] The same results were obtained with B-CLL cells (data not shown).Untreated B cells did not give rise to haematopoietic colonies (data notshown). These results therefore demonstrate the presence of viablehaemopoietic stem cells in blood samples treated with monoclonalantibody CR3/43 to the class II MHC β-chain but not in untreated bloodsamples.

[0466] Long Term Culture

[0467] The long-term assay examines the self-renewal potential ofhaematopoietic stem cells. In this culture most components of bonemarrow haematopoiesis are reproduced in vitro. The important feature ofthis culture is sustained haematopoiesis, which occurs in the absence ofadded growth factors. In this assay the process of hematopoiesis isabsolutely dependent upon the establishment of an adherent layer of bonemarrow derived stromal cells. Stromal cells (consisting of a variety ofnon-haemopoietic cells e.g., fibroblast, fat cells and including allcell types belonging to the mesenchymal system) support haematopoiesisby providing the appropriate environment (secretion of growth factorsand synthesis of extracellular matrix) to promote the survival,self-renewal, proliferation and differentiation of the stem cells.

[0468] In this assay, treatment of B cells obtained from buffy coats ofhealthy blood donors (the same results were obtained with B-CLL cells)with CR3/43 mab gave rise to the formation of an adherent cell layerwithin hours of adding the antibody which, also increased with time.

[0469] The adherent layer consisted of stromal cells (blanket cells,consisting mainly of fibroblast/mesenchymal-type cells/light refringentlarge cells when viewed with inverted bright field microscopy—see FIG.20) which supported the growth and development of haematopoietic cellsup to 12 weeks and longer (these cells show intimate contact withhaematopoietic cells). Also visible in the adherent layer are groups ofprimitive haematopoietic cells (also known as cobblestone areas/clustersof dark appearing cells) which are the origin of prolonged production ofhaematopoietic cells.

[0470] The non-adherent layers which are on top of the stromal layer(clusters of bright appearing cells) consisting of small round cellsforming clusters of haematopoietic foci. This layer contains stem cellsand also more committed progenitors of the haematopoietic system. Thenon adherent layer was capable of giving rise to MIX-CFC, GM-CFC, M-CFC,BFU-E (as determined using the clonal assay) and CFU-F (colony formingunit-fibroblast) (when sub-cultured with long term culture medium).

[0471] I. RT-PCR of Cells Treated with an Antibody to the β-Chain ofHLA-DR.

[0472] Gene transcription was measured in Ramos (B lymphoma) and K562(erythroid leukaemia) cells treated with the CR3/43 mab for the CD34,c-kit (ligand of stem cell factor), ε-haemoglobin (embryonic form ofhaemoglobin) and β-actin genes.

[0473] Methods

[0474] mRNA was extracted before and after treatment with CR3/43 mabusing RNAZOL (CINA BIOTECH). mRNA were subjected to hexamer primingreverse transcription by incubating at room temperature for 5 mins with4 μl standard buffer, 2 μl dNTPs, 1 μl RNASIN, 1 μl reverse primer(random hexamer primer) and 1 μl MMLV reverse transcriptase enzyme. Thismixture was further incubated for 1 hr at 38° C. Mixtures were thensubjected to PCR under standard conditions using primers designed toamplify CD34, c-kit, ε-haemoglobin and β-actin sequences. Primers weresynthesised at the Randell Institute Kings College according topublished data.

[0475] Results

[0476] The results obtained show that whereas the levels of β-actin mRNAdid not change, the levels of CD34, c-kit and ε-haemoglobin mRNA allincreased significantly following treatment with the CR3/43 mAb. Theresults for CD34 and c-kit provide further support for the data detailedabove that demonstrate the retrodifferentiation of B lymphocytes toproduce haemopoetic stem cells.

[0477] The results obtained for the ε-haemoglobin are even moreinteresting since ε-haemoglobin is normally only expressed in embryoniccells. It is therefore possible that treatment with the CR3/43 mAb notonly gives rise to haemopoietic stem cells but also to even moreprimitive undifferentiated cells such as embryonic stem cells.

[0478] J. Summary

[0479] In short, the examples describe in vitro experiments that revealextremely interesting, seminal findings regarding the ontogeny anddevelopment of T and B lymphocytes which can be utilised in thegeneration of stem cells to affect lymphohaematopoiesis in peripheralblood samples in a matter of hours.

[0480] Treatment of peripheral blood samples obtained from patients withB-cell chronic lymphocytic leukaemia's (B-CLL) with high B lymphocytecounts, with monoclonal antibody to the homologous region of the β-chainof class-II antigens gave rise to a marked increase in the relativenumber of single positive (SP) T lymphocytes and their progenitors whichwere double positive for the thymocyte markers CD4 and CD8 antigens andthese were coexpressed simultaneously. However, these phenomena werealways accompanied by a significant decrease in the relative number ofB-lymphocytes. These observations were not noted when the same bloodsamples were treated with monoclonal antibodies to the homologous regionof the α-chain of class-II antigens or to the homologous region ofclass-I antigens.

[0481] Treatment of whole blood obtained from patients with B-cellchronic lymphocytic leukaemia (CLL) with monoclonal antibody to thehomologous region of the B chain of the HLA-DR antigen appeared to giverise to T-lymphopoiesis. This event was marked by the appearance ofdouble positive cells coexpressing the CD4 and CD8 markers, theappearance of cells expressing CD34 and the concomitant increase in thenumber of single positive CD4⁺ CD3⁺ and CD8⁺ CD3⁺ lymphocytes.Furthermore, the immunophenotypic changes that took place in thegeneration of such cells were identical to those cited for thymocytedevelopment, especially when measured with time.

[0482] The percentages of double positive cells (DP) generated at 2 hourincubation time of whole blood with monoclonal antibody to thehomologous region of the β-chain of the DR antigen, decreased with timeand these events were accompanied by increase in the percentages ofsingle positive CD4⁺ CD3⁺ and CD8⁺ CD3 cells simultaneously and at latertimes too. TCR α and β chains were also expressed on these types ofcells.

[0483] B-lymphocytes were constantly observed to lose markers such asCD19, CD21, CD23, IgM and DR and this coincided with the appearance ofCD34⁺ and CD34⁺ CD2⁺ cells, increases in CD7⁺ cells, increases in CD8⁺CD28⁺ and CD28⁺ cells, increases in CD25⁺ cells, the appearance of CD10⁺and CD34⁺ cells and CD34⁺ and CD19⁺ cells increases in CD5⁺ cells, andcells expressing low levels of CD45 antigen. These changes were due totreatment of blood with monoclonal antibody to the homologous region ofthe β-chain of HLA-DR antigen.

[0484] The immunophenotypic changes associated with such treatment isconsistent with retrodifferentiation and subsequent commitment (i.e.recommitment) of B lymphocytes, because the majority of white bloodcells in blood of patients with B-CLL before treatment were Blymphocytes. Furthermore, B-lymphocytes of patients with B-CLL whichwere induced to become T-lymphocytes following treatment withcyclophosphamide and monoclonal antibody to the β-chain of HLA-DRantigen, were able to revert back to B lymphocytes following prolongedincubation with this treatment.

[0485] On analysis of treated samples with monoclonal antibody to theβ-chain of HLA-DR antigen, with CD16&56 and CD3 and CD8 and CD3 panels,the relative number of cells expressing these markers steadily increasesin increments consistent with those determined with panels such as CD19and CD3 and DR and CD3. Investigation of the supernatant of treated anduntreated samples of patients with HIV infection using nephlometry andimmunoelectrophoresis reveals increased levels of IgG indicating thatthe B-cells must have passed through the plasma cell stage. The increasein the relative number of all above-mentioned cells was also accompaniedby the appearance of medium size heavily granulated cells expressing theCD56&16 antigens in extremely high amounts. Other cells which wereextremely large and heavily granulated were observed transiently andthese were positive for CD34 and double positive for CD4 CD8 markers.Other transient cells were also observed and these were large andgranular and positive for the CD3 and CD19 receptors. CD25 which waspresent on the majority of B-lymphocytes was lost and became expressedby newly formed T-lymphocytes which were always observed to increase innumber.

[0486] CD28⁺CD8⁺ and CD28⁺ cells appeared after treatment of whole bloodof patients with B-CLL with monoclonal antibody to the homologous regionof the B chain of the DR antigen. These findings were due to treatmentof blood with monoclonal antibody to the homologous region of theβ-chain of HLA-DR antigen.

[0487] T-lymphopoiesis generated in this manner was also observed inperipheral blood of healthy blood donors, cord blood, bone marrow,patients with various infections including HIV⁺ individuals and AIDSpatients, enriched fractions for B lymphocytes obtained from bloodsamples of healthy blood donors, IgA deficient patients and otherpatients with various other conditions. Furthermore, analysis of myeloidmarkers in treated samples of two patients with B-CLL with monoclonalantibody to the homologous region of the β-chain of the HLA-DR antigenshowed a significant increase in the relative number of cells expressingthe myeloid markers such as CD13 and CD33. These markers werecoexpressed with the CD56 & 16 or the CD7 antigens. However, therelative number of CD7⁺ cells with T-lymphocyte markers and withoutmyeloid antigens was observed on a separate population of cells. Theseparticular observations were not seen in untreated samples or in samplestreated with monoclonal antibodies to class I antigens or the homologousregion of the α-chain of HLA-DR antigen (see Charts 2 & 3). These finalresults suggest that B-lymphocytes once triggered via the β-chain of theHLA-DR antigen are not only able to regress into T lymphocyte progenitorcells but are also capable of existing into the myeloid and erythroidlineages.

[0488] Thus in summary, the data presented in the present applicationdemonstrate that (i) it is possible to convert healthy cells from onelineage to cells having the cell surface markers and morphologicalcharacteristics of cells of several other lineages and (ii) it ispossible to obtain cells having the cell surface markers andmorphological characteristics of primitive precursor cells (for examplestem cells), from differentiated B lymphocytes and T lymphocytes.

[0489] It should be noted that a number of experiments have been carriedout with BCLL cells. BCLL cells are mature B lymphocytes that areincapable of differentiating to the final terminally differentiatedstage of a plasma cell. Instead, due to a chromosome defect, theyexhibit high levels of proliferation, hence the large numbers of Blymphocytes in the blood of BCLL patients. By contrast to a number oftumour cells described in the prior art, BCLL cells have not undergoneany form of limited reverse differentiation prior to use in the methodsof the invention. Furthermore they do not exhibit any characteristics ofundifferentiated cells in term of genomic structure, cell markers orcell morphology. They are in all respects mature B lymphocytes.

[0490] Thus whereas some malignant cells may have to a limited extentsome characteristics of undifferentiated cells, this is not the case forBCLL cells, which are a perfectly acceptable experimental system forstudying B lymphocytes. In fact BCLL and Daudi cells are notsufficiently distinguished from normal cells in any aspects relevant tothese experiments. Indeed, the suitability of BCLL cells as a modelsystem is confirmed by Martensson et al., 1989, Eur. J. Immunol. 19:1625-1629 (see page 1625 rhs, 1^(st) para).

[0491] It should be noted that the stem cells that are produced by themethod of the present invention may be stem cells of any tissue and arenot necessarily limited to lymphohaematopoietic progenitor cells.

[0492] Other modifications of the present invention will be apparent tothose skilled in the art. TABLE 1 CLINICAL DIAGNOSIS OF PATIENTS ANDEXPERIMENTAL CONDITIONS OF BLOOD SAMPLES INCLUDING COULTER COUNTS (WBC)FOLLOWING AND PRIOR TREATMENT OF BLOOD SPECIMENS WITH VARIOUS MONOCLONALANTIBODIES AND OTHER AGENTS WBC/L #LYMPH/L PATIENT EXPT X10-9 % LYMPH10X-9 AGENT ID DIAGNOSIS COND B A B A B A ML/mL  1 B-CLL 12 HR 100 ND86.1 ND 86.1 ND ANTI-B AT 22 C. 50  2 B-CLL 2 HR 39.1 9.6 74.4 63.3 29.96.1 ANTI-B AT 22 C. 50 2 HR 39.1 37.7 74.4 75.1 29.9 28.3 ANTI-B AT 22C. PE 50  3 B-CLL 6 HR 39.5 9.3 71.9 67.2 28.3 6.2 ANTI-B AT 22 C. 50 6HR 39.5 37.7 71.9 72.5 28.3 27.4 ANTI-B AT 22 C. PE 50  4 B-CLL 24 HR 399.3 73 66.5 28.4 6.2 ANTI-B AT 22 C. 50 24 HR 39 36.2 73 70.4 28.4 25.5ANTI-B AT 22 C. PE 50  5 B-CLL 2 HR ANTI-B AT 22 C. 50 ANTI-A 50 ANTI-I50 ANTI-B & TOXIC AGENT 25 + 25  6 B-CLL 24 HR ANTI-B AT 22 C. 50  7B-CLL 24 HR 170 128 95.4 91.1 16.9 11.6 ANTI-B AT 22 C. 178 94.2 16.8 10130 90.4 11.9 ANTI-I 10 ANTI-B & TOXIC AGENT 10 + 20  8 B-CLL 24 HR 16 781.9 51.2 14 3.0 ANTI-B AT 22 C. 20  9 B-CLL 12 HR +++ 89.5 87 85.1 +++76.2 ANTI-B AT 22 C. 85.4 30 +++ +++ ANTI-I 89.4 30 +++ 84.9 +++ ANTI-495.4 30 ANTI-I + II + 4 10 + 10 + 10 10 B-CLL 2 HR 19.3 ND 86 ND 16.7 NDANTI-B AT 22 C. 30 ANTI-I 30 92 OUT 2 HR 5.4 ND 74.5 ND ND ANTI-BPATIENT AT 22 C. 20 87 OUT 2 HR 4.8 ND 59.3 ND ND ANTI-B PATIENT AT 22C. 20 91 OUT 2 HR 4.2 ND 54.0 ND ND ANTI-B PATIENT AT 22 C. 20 21 OUT 2HR 3.9 ND 47.4 ND ND ANTI-B PATIENT AT 22 C. 20 34 OUT 2 HR 7.2 ND 20.0ND ND ANTI-B PATIENT AT 22 C. 20 36 CMV 4 HR 13.4 ND 7.3 ND ND ANTI-BINFANT AT 22 C. 20 93 HIV+ 4 HR 5.6 ND 43.4 ND ND ANTI-B INFANT AT 22 C.20 BB/ST 40% BLAST 2 HR 60.5 ND 20.2 ND 12.2 ND ANTI-B IN BLOOD AT 22 C.50 6 DAYS 24 HR ANTI-A OLD AT 22 C. 50 ANTI-AB 25 + 25 HIV25 AIDS 2 HR7.5 ND 34.8 ND 2.6 ND ANTI-B AT 22 C. 50 ANTI-A 50 ANTI-AB 25 + 25 43/BDB CELL 4 HR ANTI-B DEFICIENT AT 22 C. 20 ANTI-I 20 ANTI-4 20 OB/BD BCELL 4 HR ANTI-B DEFICIENT AT 22 C. 20 ANTI-I 20 ANTI-4 20 HIV+ AIDS 6HR ANTI-B AT 22 C. 20 ANTI-I IgA-D IgA 6 HR ANTI-B DEFICIENT AT 22 C. 20ANTI-I 20

[0493] TABLE 2 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITH CD19AND CD3 MONOCLONAL ANTIBODIES. % CD19 + % CD19 + % CD3 − HGCD3 − % CD19+% CD3+ CD3+ CD19− FC+ PATIENT B A B A B A B A B A 1 88 40 5 19 1 2 6 260 12 2 73 15 10 33 2 7 15 41 0 5 3 73 11 11 33 2 2 14 52 0 2 4 71 13 1137 2 2 16 47 0 2 5 85 40 5 16 1 1 6 26 3 18 6 85 43 5 18 1 1 6 27 3 10 790 72 2 4 0 2 7 8 0 14 8 62 25 7 13 0 1 29 55 2 6 9 90 85 2 3 0 0 2 1 14 10  78 50 7 14 0 0 14 26 0 8 92  12 10 38 49 0 1 49 40 0 0 91  7 3 3529 0 1 59 67 0 0 87  5 3 32 38 1 1 63 58 0 0 21  1 1 27 29 1 0 71 70 0 034  1 1 13 13 0 2 86 84 0 0 39  10 6 23 25 0 0 67 69 0 0 93  6 3 26 27 11 68 70 0 0 BB/ST 1 1 12 13 0 0 87 86 0 0 HIV25 7 2 26 27 0 0 68 67 0 043/BD 0 0 40 42 0 1 58 54 0 0 04/BD 0 0 49 41 0 3 43 41 0 0 HIV+ 1 1 1014 0 0 89 87 0 0 IgA/D 10 1 21 25 2 3 67 71 0 0

[0494] TABLE 3 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONALANTIBODY TO THE B CHAIN OF THE HOMOLOGOUS REGION OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD4 AND CD8. % CD8+ % CD4+ % CD4 + CD8+ % CD4 −CD8− CD4 + LOW PATIENT B A B A B A B A B A 1 2.8 16 2.9 11.4 0 3.2 93.167.6 0 0 2 6.2 13.2 9.1 24.3 0 9.4 78.7 46 5.8 6.3 3 7.2 13.1 7.4 23.9 08.2 78.8 48.1 6.3 6.6 4 10.1 24.2 7.6 24.9 0.3 2.8 77.5 42 4.6 5 5 2.916.2 1.8 7.6 0 2 95 62.3 0 0 6 ND 12 ND 8.1 ND 1.7 ND 75.7 ND 0 7 1.92.6 1.9 2.8 0 0 95.8 94.3 0 0 8 3.2 7 3.9 6.9 0.1 2 87.3 79.8 4.3 6 92.8 2.9 3 3 0 0 94 94.1 0 0 10  5.7 9.4 4.7 9.1 0.6 0.8 88.7 79.2 0 092  21 19 21.6 21 0.8 1.9 50.5 52.5 5.3 4.8 91  15.4 18.1 13.6 17.9 6.22.6 57 57.3 7.3 3.5 87  16.8 21.8 13.4 20.4 2.9 2.6 59.5 48.9 7 5.6 21 16 24.1 9.1 15.2 1 2.6 69.6 53.2 3.7 4.2 34  9.4 11.9 5.7 4.9 2 3.3 67.665.3 14.4 14.5 39  12.1 12.6 13.1 14.6 0.4 1.3 62.3 66.7 11.9 4.3 93 18.9 20.3 9.7 10.3 1.8 1.4 65.5 65.9 3.4 1.8 BB/ST 6.3 13 5.7 7.3 2.21.1 34.7 70.3 50.3 7.6 HIV25 24.1 24.9 0.8 1.1 1.3 5 70.2 69.3 2.9 3.8

[0495] TABLE 4 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF SAMPLES WITH MONOCLONALANTIBODY TO THE B CHAIN OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD3AND DR DR+ CD+ CD + DR+ DR − CD3− DR + HCD3− PATIENT B A B A B A B A B A1 87   45.5 3.5 20.8 2.5 4.2 6.9 21.6 0 7.6 2 76.2 19.4 9.6 29.2 3.9 8.710.3 36.8 0 5.5 3 77.7 18.3 8.4 29.4 4.1 8.8 9.6 38.1 0 4.7 4 76.8 19.27.6 29.5 6.2 10.5 9.1 37.2 0 3.3 5 ND 47.1 ND 11.5 ND 9.9 ND 22.4 ND 7.36 ND 7 91.4 85.8 2.4 2.5 0.7 0.7 5.1 4.2 0 6.3 8 61.8 28.9 6.5 11.2 23.3 28.6 54.6 0 1.5 9 ND 10  82.6 44.7 4.3 9.8 3.3 5 9.8 22.2 0 17.9 92 23.8 14.1 39.3 41.9 4.5 3.5 32.4 40.5 0 0 91  13.3 7.9 29.6 32.5 3.4 2.953.4 56.5 0 0 87  14.8 12.2 28.4 34.1 5.5 6.6 51.1 46.5 0 0 21  ND 34 11.9 12.9 10.4 13.7 0.8 0.6 76.7 72.8 0 0 39  25.6 13.7 24.6 25.2 3 2.846.5 25.2 0 0 93  13.3 8.9 18.4 18.9 9.9 10.1 58.2 61.7 0 0 BB/ST 44.232.5 11.7 12.2 0.8 0.8 43 49.4 0 4.6

[0496] TABLE 5 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD16 + 56 AND CD3. PA- CD56 + &16 CD3+ CD56 +&16 + CD3+ CD56 + &16 − CD3− TIENTS B A 8 A B A B A 1 2 4.3 5.7 19.7 0.71.7 91.3 73 2 11.5 38.9 12.4 32.6 1 6.6 74.5 21 3 12 36.2 12.1 34.5 0.76 75.5 23 4 12.2 32.6 12.4 39.6 0.5 5 74.7 22.2 5 ND 13.1 ND 9.4 ND 2.6ND 73.5 6 ND 7 0.8 0.8 2.8 2.4 0.3 0.2 96.2 96.4 8 24.8 52 5.4 12.4 0.94.1 68.3 31.1 9 ND 10  1.1 1.3 6.1 13.7 2.1 2.5 90.5 82.4 92  23.8 34.544.3 44.8 2 1.5 29.2 18.6 91  4.6 3.9 28.8 29.4 3 3.2 63.3 63.3 87  47.946.4 28.8 36.5 5.8 3.7 16.9 13 21  9.4 9.4 19.7 23.6 4.2 6.7 66   59.534  21.5 12.8 11.4 13.7 1.8 0.6 64.6 72.8 39  7 2.7 23.4 26.1 1.1 0.168.2 71 93  55.8 54.9 26.2 26.3 1.7 2 16.1 16.8 BB/ST 28.8 29.9 12 14.30.8 1.8 49.4 53.6

[0497] TABLE 6 IMMUNOPHENTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER OF TREATMENT OF BLOOD WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD45 AND CD14. CD45 + H CD45 + L CD45 + CD14+PATIENTS B A B A B A 1 90.5 70.1 7.5 21.9 0.8 3.3 2 65.8 52.2 8.8 38.35.3 9.5 3 84.3 52.2 9.9 33.8 5.1 13.2 4 91.5 79.2 2.1 7 5.7 10.8 5 63.184.6 34.9 9.4 0.5 3.6 6 ND 7 52.8 85.2 45.6 13.9 0.5 0.6 8 71.1 55 71.134.5 5.3 8.7 9 SEE 10  79.7 47.3 16.3 48 2.1 1.9 92  61.7 64.7 27.4 26.65.9 3.6 91  49.4 49.2 40.4 44.3 6.5 3.2 87  52.4 61.5 36.1 28.7 7 6.521  45.8 43.3 44.3 47.6 6.2 3.3 34  24.4 24.6 54.8 59.6 13.3 9.7 39 48.7 46.3 30.5 42.1 14.5 8.8 93  SEE HIV+ 22.6 26.9 66.8 63.5 6.8 6.7IgA/D 47.4 59.8 41.9 33.3 5.9 4.1

[0498] TABLE 7 IMMUNOPHENOTYPING OF PATIENT WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD WITH MONOCLONALANTIBODIES TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD8 AND CD3. CD8+ CD3+ CD8 + CD3+ CD8 − CD3−PATIENTS B A B A B A B A  2 0.6 1.3 7.5 19.3 4.2 19.3 87.7 63.8  3 1.11.4 8.3 20.3 5.6 18.4 84.8 59.8  4 3.5 2.9 8.3 27 3.9 18.6 84.2 53.1 923.5 1.9 27.6 25.2 18.4 19 50.3 52.8 91 4 3.1 18.2 19 14.1 12.6 63.6 65.387 5.7 3.9 19.9 23.6 15.4 17.4 58.8 55 21 4.8 7.4 16.3 17.3 13.7 13 65.262 34 3 3.6 5.2 6.7 7.6 7.5 84.1 82.3

[0499] TABLE 8 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURE WITH MONOCLONALANTIBODIES TO CD45 AND CD14. TIME DR + CD45 + CD14 + r CD45 + L CD45 + H 2 HR 81.7 8.2 8.2  6 HR 80.7 8.1 10.6 24 HR 79 1.1 18.4

[0500] TABLE 9 IMMUNOPHENOTYPING OF A PATINENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD19 AND CD3. CD19 + CD3 + CD19 − TIME DR + r CD3+ DR+ CD3− DR−  2 HR 87.4 10.1 1.8 10.7  6 HR 75.5 10.4 31 10.7 24 HR 74 11.7 2.911

[0501] TABLE 10 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD4 AND CD8. CD8 + CD4 + &CD8 + CD4 + CD4 − TIME & DR + rCD4+ &DR + r DR+ CD8 − DR− 2 HR 77.6 6.8 5.4 1.3 8.8 6 HR 75.8 6.7 6.41.8 9.3 24 HR  77 6.4 4.8 1.9 11

[0502] TABLE 11 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD3 AND DR. TIME DR+ CD3+ CD3 + DR+ CD3 + DR−  2 HR 75 9.54.2 10.9  6 HR 74.8 8.8 4.8 10.9 24 HR ND ND ND ND

[0503] TABLE 12 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD16&56 AND CD3. TIME CD56 + &16 + DR + r CD3+ CD56 +CD16 + &CD3 + DR + r CD56 − CD16 − &CD16 − DR−  2 HR 82.5 9.5 4.1 3.5  6HR 84.3 7.5 4.1 3.3 24 HR ND ND ND ND

[0504] TABLE 13 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD8 AND CD3. CD8 + CD8 + CD + CD8 − CD3 − TIME DR+ CD3+3&DR + r DR− 2 HR 76.2 6.6 6.7 10.6 6 HR 76.5 6.2 6.2 10.3

[0505] TABLE 14 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL BEFORE ANDAFTER TREATMENT OF BLOOD WITH MONOCLONAL ANTIBODIES TO THE HOMOLOGOUSREGION OF THE A-CHAIN OF THE HLA-DR, THE HOMOLOGOUS REGION OF THEB-CHAIN OF THE HLA-DR, THE TWO MONOCLONAL TOGETHER, MONOCLONAL TO THEHOMOLOGOUS REGION OF THE B-CHAIN PLUS CYCLOPHOSPHOAMIDE AND THEHOMOLOGOUS REGION OF CLASS I ANTIGENS MEASURED WITH TIME. CD19+ CD3+CD19 + CD3+ CD19 − CD3− ID B AA AB ABC AI B AA AB ABC AI B AA AB ABC AIB AA AB ABC AI 5/6 2 H 86 91 54 40 89 5 4 16 23 5 1 1 3 2 1 6 4 27 33 524 N 88 51 60 86 N 4 18 10 4 N 2 1 2 3 N 4 29 28 7 10 2 H 77 N 59 N 80 7N 13 N 7 1 N 1 N 0 14 N 26 N 12 09 24 8 N N N 6 32 N N N 38 1 N N N 1 59N N N 56 43/BD 6 H 0 N 0 0 0 40 N 42 43 49 0 N 1 0 1 58 N 54 54 47 04/BD6 H 0 N 0 0 0 49 N 41 45 46 0 N 3 1 3 43 N 42 44 41 HIV+ 6 H 1 N 0 N 110 N 14 N 12 0 N 0 N 0 89 N 86 N 87 IgA/D 6 H 10 N 1 N 12 21 N 25 N 20 2N 1 N 3 67 N 71 N 68

[0506] TABLE 15 CD8 AND CD4 Error! Bookmark not defined. CD19+ CD3+CD19 + CD3+ CD19 − CD3− ID B AA AB ABC AI B AA AB ABC AI B AA AB ABC AIB AA AB ABC AI 5/6 2 H 3 2 14 10 4 2 2 8 8 3 0 0 3 2 1 95 94 74 79 93 24N 3 9 4 4 N 3 8 4 3 N 0 2 2 0 N 94 81 90 93 10 2 H 3 N 7 N 4 4 N 7 N 3 1N 2 N 1 91 N 83 N 92 09 24 10 N N N 15 21 N N N 38 2 N N N 2 61 N N N 53

[0507] TABLE 16 DR+ CD3+ CD3+DR+ CD3−DR− A A A A A A A A ID B A AB BC AlB A AB BC Al B A AB BC Al B A AB BC Al 5/6 N 90 54 N 87 N 4 12 N  4 N 210 N 3 N 5 22 N  5 2H 10 83 N 63 N 81  4 N  8 N  4 4 N  7 N 4  9 N 23 N12 2H 09 14 N N N 13 30 N N N 36 3 N N N 3 51 N N N 47 24

[0508] TABLE 17 CD16&56 AND CD3 CD56+&16+ CD3+ CD56+&16+CD3+CD56−&16−CD3− A A A A A A A A ID B A AB BC Al B A AB BC Al B A AB BC AlB A AB BC Al 5/6 N 0 13 N  4 N 5  9 N  5 N 1 3 N 1 N 94 74 N 90 2H 10  0N  1 N  1  6 N 14 N  6 1 N 2 N 1 92 N 65 N 92 2H 09 42 N N N 41 36 N N N38 2 N N N 2 20 N N N 19 24

[0509] TABLE 18 CD45 AND CD14 CD45+L CD45+M CD45+H CD45+CD14+ A A A A AA A A ID B A AB BC Al B A AB BC Al B A AB BC Al B A AB BC Al 5/6 0 0 510 0 44 43 50 50 32 55 43 50 31 67 1 1 1 2 0 2H 10 0 N 0 N 0 43 N 54 N35 54 N 42 N 62 1 N 1 N 0 2H 09 2 N N N 1 18 N N N 16 71 N N N 76 7 N NN 5 24 HIV + 4 N 3 N 6 63 N 61 N 41 23 N 27 N 40 7 N 7 N 7 6H lgA/ 2 N 2N 4 40 N 31 N 44 47 N 60 N 44 6 N 4 N 6 D 6H

[0510] TABLE 19 CD8 AND CD28 CD8+ CD28+ CD8+CD28+ CD8−CD28− A A A A A AA A ID B A AB BC Al B A AB BC Al B A AB BC Al B A AB BC Al 5/6 N 3 6 N 3N 1 4 N 2 N 1 4 N 1 N 95 86 N 94 2H 8 4 N 6 N N 3 N 5 N N 1 N 3 N N 92 N86 N N 2H

[0511] TABLE 20 CD34 AND CD2 CD34+ CD2+ CD34+CD2+ CD34−CD2− A A A A A AA A ID B A AB BC Al B A AB BC Al B A AB BC Al B A AB BC Al 5/6 2H N  134 N N N  6 13 N N N  3 30 N N N 90 21 N N 24 N  1  6  9 N N  7 23  4 NN  3 33 43 N N 87 34 34 N HIV +  2  1 12 13 N 20 21 21 12 N  4  5  9 14N 73 73 64 60 N 2H BB/ ST 2H 26 23 33 14 N 15 14 15 15 N 31 30 23 36 N27 32 28 35 N 24 N 11 29 11 N N 13 12  9 N N 27  9 18 N N 48 49 61 N

[0512] CHART 1 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODSAMPLE OF PATIENT (2, 3 & 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION OF THE β-CHAIN OF HLA-DR ANTIGEN MEASURED WITH TIME. WITHOUT WITHFL1 FL2 TIME NOTHING001 WITH002 CD45 CD14  2 HR NO001 WE002 CD45 CD14  6HR 001001 002002 CD45 CD14 24 HR NOTHING003 WITH004 CD3 CD19  2 HR NO003WE004 CD3 CD19  6 HR 001003 002004 CD3 CD19 24 HR NOTHING004 WITH005 CD4CD8  2 HR NO004 WE005 CD4 CD8  6 HR 001004 002005 CD4 CD8 24 HRNOTHING005 WITH006 CD3 DR  2 HR NO005 WE006 CD3 DR  6 HR 001005 002006CD3 DR 24 HR NOTHING006 WITH007 CD3 CD56&16  2 HR NO006 WE007 CD3CD56&16  6 HR 001006 002007 CD3 CD56&16 24 HR N003 W004 CD3 CD8  2 HRNO007 WE008 CD3 CD8  6 HR 001007 002008 CD3 CD8 24 HR

[0513] CHART 1A IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODSAMPLE OF PATIENT (2, 3, 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION OF THE β-CHAIN OF HLA-DR ANTIGEN CONJUGATED TO PE MEASURED WITHTIME. ID FL1 FL2 TIME WL003 CD45 CD14  2 HR WEL003 CD45 CD14  6 HR003003 CD45 CD14 24 HR WL005 CD3 CD19  2 HR WEL005 CD3 CD19  6 HR 003005CD3 CD19 24 HR WL006 CD4 CD8  2 HR WEL006 CD4 CD8  6 HR 003006 CD4 CD824 HR WL007 CD3 DR  2 HR WEL 007 CD3 DR  6 HR WL008 CD3 CD65&16  2 HRWEL 008 CD3 CD56&16  6 HR WL005 CD3 CD8  2 HR WEL009 CD3 CD8  6 HR

[0514] CHART 2 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODOF PATIENT (1) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN, THIS ANTIBODY AND CYCLOPHOSPHAMIDE,MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE α-CHAIN OF HLA-DRANTIGEN AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF CLASS IANTIGEN MEASURED WITH TIME. Error! WITH- Bookmark WITH OUT FL1 FL2 TIMENA001 CD45 CD14  2 HR A2B001:AB CD45 CD14  2 HR A2A:AA CD45 CD14  2 HRDNAA001:ABC CD45 CD14  2 HR A1001:AI CD45 CD14  2 HR NC001 CD3 CD19  2HR C2B001:AB CD3 CD19  2 HR C2A001:AA CD3 CD19  2 HR DNAC001:ABC CD3CD19  2 HR C1001:AI CD3 CD19  2 HR A124H001:AI CD3 CD19 24 HRA2B24H001:AB CD3 CD19 24 HR A2A24H001:AA CD3 CD19 24 HR A2BX24H001:ABCD3 CD19 24 HR ND001 CD4 CD8  2 HR D2B001:AB CD4 CD8  2 HR D2A001:AA CD4CD8  2 HR DNAD001:ABC CD4 CD8  2 HR D1001:AI CD4 CD8  2 HR D124H001:AICD4 CD8 24 HR D2BX24H001:AB CD4 CD8 24 HR D2B001:AB CD4 CD8 24 HRD2A001:AA CD4 CD8 24 HR E1001:AI CD3 DR  2 HR E2B001:AB CD3 DR  2 HRE2A001:AA CD3 DR  2 HR F1001:AI CD3 CD56&16  2 HR F2B001:AB CD3 CD56&16 2 HR F2A001:AA CD3 CD56&16  2 HR G1001:AI CD28 CD8  2 HR G2A001:AA CD28CD8  2 HR G2B001:AB CD28 CD8  2 HR H1001:AI CD7 CD33&13  2 HR H2A001:AACD7 CD33&13  2 HR H2B001:AB CD7 CD33&13  2 HR I2A001:AA CD21 CD5  2 HRI2B001:AB CD21 CD5  2 HR J2A001:AA CD34 CD2  2 HR J2B001:AB CD34 CD2  2HR B2A24H001:AA CD34 CD2 24 HR B2B24H001:AB CD34 CD2 24 HRB2BX24H001:ABC CD34 CD2 24 HR K2B001:AB CD10 CD25  2 HR K2A001:AA CD10CD25  2 HR

[0515] CHART 3 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODOF PATIENT (8) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN. WITH WITHOUT FL1 FL2 TIME AN001 CD45 CD14 2HR A2001 CD45 CD14 2 HR CN001 CD3 CD19 2 HR C2001 CD3 CD19 2 HR DN001CD4 CD8 2 HR D2001 CD4 CD8 2 HR EN001 CD3 DR 2 HR E2001 CD3 DR 2 HRFN001 CD3 CD56&16 2 HR F2001 CD3 CD56&16 2 HR GN001 CD28 CD8 2 HR G2001CD28 CD8 2 HR HN001 CD7 CD5 2 HR H2001 CD7 CD5 2 HR IN001 CD13 CD20 2 HRI2001 CD13 CD20 2 HR JN001 CD45RA CD25 2 HR J2001 CD45RA CD25 2 HR KN001CD57 CD23 2 HR K2001 CD57 CD23 2 HR

[0516] CHART 4 IIMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODSAMPLE OF PATIENT (10) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE β-CHAIN OF HLA-DR ANTIGEN AND MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF CLASS 1 ANTIGENS. WITH WITHOUT FL1 FL2 TIME CLL0001CD45 CD14 2 HR CLL1001 CD45 CD14 2 HR CLL2001 CD45 CD14 2 HR CLL0003 CD3CD19 2 HR CD3 CD19 2 HR CLL1003 CD3 CD19 2 HR CLL2003 CD3 CD19 2 HRCLL0004 CD4 CD8 2 HR CLL1004 CD4 CD8 2 HR CLL2004 CD4 CD8 2 HR CLL005CD3 DR 2 HR CLL1005 CD3 DR 2 HR CLL2005 CD3 DR 2 HR CLL0006 CD3 CD56&162 HR CLL1006 CD3 CD56&16 2 HR CLL2006 CD3 CD56&16 2 HR

We claim
 1. A method of preparing an undifferentiated cell, the methodcomprising contacting a more committed cell with an agent that causesthe more committed cell to retrodifferentiate into an undifferentiatedcell.
 2. The method according to claim 1 wherein the committed cells arenon-cancer cells.
 3. The method according to claim 1 wherein thecommitted cells are differentiated cells.
 4. The method according toclaim 3 wherein the committed cells are selected from CFC-T cells, CFC-Bcells, CFC-Eosin cells, CFC-Bas cells, CFC-GM cells, CFC-MEG cells,BFC-E cells, CFC-E cells, T cells and B cells.
 5. The method accordingto claim 1 wherein the undifferentiated cells are pluripotent stemcells.
 6. The method according to claim 1 wherein the undifferentiatedcells are stem cells selected from the group consisting of haemopoieticstem cells, neuronal stem cells, epithelial stem cell, mesenchymal stemcells and embryonic stem cells.
 7. The method according to claim 1wherein the undifferentiated cells are MHC class I⁺ and/or MHC class II⁺cells.
 8. The method according to claim 1 wherein the agent engages areceptor that mediates capture, recognition or presentation of anantigen at the surface of the committed cells.
 9. The method accordingto claim 8 wherein the receptor is an MHC class I antigen or an MHCclass II antigen.
 10. A method according to claim 9 wherein said class Iantigen is an HLA-A receptor, an HLA-B receptor, an HLA-C receptor, anHLA-E receptor, an HLA-F receptor or an HLA-G receptor and said class IIantigen is an HLA-DM receptor, an HLA-DP receptor, an HLA-DQ receptor oran HLA-DR receptor.
 11. The method according to claim 10 wherein thereceptor is an HLA-DR receptor.
 12. The method according to claim 8wherein the receptor comprises a β-chain having homologous regions. 13.The method according to claim 12 wherein the receptor comprises at leastthe homologous regions of the β-chain of HLA-DR.
 14. The methodaccording to claim 8 wherein the agent is an antibody to the receptor.15. A method according to claim 14 wherein the agent is a monoclonalantibody to the receptor.
 16. A method according to claim 15 wherein theantibody is selected from the group consisting of monoclonal antibodyCR3/43 and monoclonal antibody TAL 1B5.
 17. A method according to claim1 wherein the agent modulates MHC gene expression.
 18. A methodaccording to claim 17 wherein the agent modulates MHC Class I⁺ and/orMHC Class II⁺ expression.
 19. A method according to claim 1 wherein theagent is used in conjunction with a biological response modifier.
 20. Amethod according to claim 19 wherein the biological response modifier isselected from the group consisting of an alkylating agent, animmunomodulator, a growth factor, a cytokine, a cell surface receptor, ahormone, a nucleic acid, a nucleotide sequence, an antigen and apeptide.
 21. A method according to claim 20 wherein the alkylating agentis or comprises cyclophosphoamide.
 22. A method according to claim 1wherein the method is an in vitro method.
 23. A method according toclaim 1 further comprising recommitting the undifferentiated cells intomore differentiated cells.
 24. A method according to claim 23 whereinthe more differentiated cells are of the same lineage as the committedcells.
 25. A method according to claim 23 wherein the moredifferentiated cells are of a different lineage to the committed cells.26. An undifferentiated cell produced according to the method ofclaim
 1. 27. A recommitted cell produced according to the method ofclaim
 23. 28. The method according to claim 1 further comprisingenriching said undifferentiated cells or recovering saidundifferentiated cells from the cell population by using a cell surfacemarker.
 29. The method according to claim 28 wherein saidundifferentiated cells are recovered from the cell population after 2hours.
 30. A method of producing an altered cell population comprisingundifferentiated cells capable of being recommitted into moredifferentiated cells, which method comprises: (i) contacting an initialcell population comprising committed cells with an agent that operablyengages said committed cells; and (ii) culturing committed cells withinthe cell population that are engaged by said agent to becomeundifferentiated cells as a result of said engaging, thereby resultingin an altered cell population comprising said undifferentiated cells;and optionally (iii) enriching said undifferentiated cells or recoveringsaid undifferentiated cells from the altered cell population.
 31. Amethod of producing undifferentiated cells from committed cells, saidundifferentiated cells being capable of being recommitted into moredifferentiated cells, which method comprises: (i) contacting an initialcell population comprising the committed cells with an agent thatoperably engages the surface of said differentiated cells; and (ii)culturing committed cells that are engaged by said agent to becomeundifferentiated cells as a result of said engaging, thereby producingundifferentiated cells from committed cells and resulting in an alteredcell population comprising said undifferentiated cells; and optionally(iii) enriching said undifferentiated cells or recovering saidundifferentiated cells from the altered cell population.
 32. The methodaccording to claim 31 wherein step (iii) comprises enriching saidundifferentiated cells or recovering said undifferentiated cells fromthe altered cell population by using a cell surface marker.
 33. Themethod according to claim 31 wherein said undifferentiated cells arerecovered from the altered cell population after 2 hours.
 34. A methodof inducing in a cell population committed cells of one hemopoieticlineage to become cells of another lineage which method comprises: (i)contacting the cell population with an agent that operably engages saidcommitted cells; and (ii) culturing committed cells that are engaged bysaid agent to become cells of another lineage as a result of saidengaging.
 35. A method according to claim 34 wherein said committedcells are of a B cell lineage and become cells of another hemopoieticlineage selected from a T cell lineage and a myeloid lineage.
 36. Amethod of producing T cells from B cells which method comprises (i)contacting an initial cell population comprising B cells with an agentthat engages the β chain of an MHC class II antigen present on thesurface of said B cells; and (ii) culturing B cells that are engaged bysaid agent to become T cells as a result of said engaging, therebyresulting in T cells from B cells and an altered cell populationcomprising said T cells; and optionally (iii) enriching said T cells orrecovering said T cells from the altered cell population.
 37. A methodof producing myeloid cells from B cells which method comprises (i)contacting an initial cell population comprising B cells with an agentthat engages the β chain of an MHC class II antigen present on thesurface of said B cells; and (ii) culturing B cells that are engaged bysaid agent to become myeloid cells as a result of said engaging, therebyresulting in myeloid cells from B cells and an altered cell populationcomprising said myeloid cells; and optionally (iii) enriching saidmyeloid cells or recovering said myeloid cells from the altered cellpopulation.
 38. A method of producing stem cells from B cells, whichmethod comprises: (i) contacting an initial cell population comprising Bcells with an agent that engages the β chain of an MHC class II antigenpresent on the surface of said B cells; and (ii) culturing B cells thatare engaged by said agent to become stem cells as a result of saidengaging, thereby resulting in stem cells from B cells and an alteredcell population comprising said stem cells; and optionally (iii)enriching said stem cells or recovering said stem cells from the alteredcell population.
 39. A method of inducing in a cell population a loss ofappearance of one or more cell surface markers of committed cellsfollowed by appearance of one or more cell surface markers of stemcells, which method comprises: (i) contacting the cell population withan agent that operably engages said committed cells; and (ii) culturingcommitted cells that are engaged by said agent to have appearance of oneor more cell surface markers of stem cells as a result of said engaging.40. The method of claim 39 wherein the committed cells are hemopoietic.41. The method of claim 39 wherein the agent engages a receptor thatmediates capture, recognition or presentation of an antigen at thesurface of the committed cells.
 42. The method according to claim 39further comprising enriching said stem cells or recovering said stemcells from the cell population by using a cell surface marker.
 43. Amethod of producing an altered cell population comprising cells havingan appearance of one or more cell surface markers of stem cells, whichmethod comprises: (i) contacting an initial cell population comprisingcommitted cells with an agent that operably engages said committedcells; and (ii) culturing committed cells within the cell populationthat are engaged by said agent to become cells having an appearance ofone or more cell surface markers of stem cells as a result of saidengaging, thereby resulting in an altered cell population comprisingsaid stem cells; and optionally (iii) enriching said stem cells orrecovering said stem cells from the altered cell population.
 44. Amethod of producing cells having an appearance of one or more cellsurface markers of stem cells capable of being recommitted into moredifferentiated cells, which method comprises: (i) contacting an initialcell population comprising committed cells with an agent that operablyengages the surface of said differentiated cells; and (ii) culturingcommitted cells that are engaged by said agent to become cells having anappearance of one or more cell surface markers of stem cells as a resultof said engaging, thereby producing cells having an appearance of one ormore cell surface markers of stem cells capable of being recommittedinto more differentiated cells and resulting in an altered cellpopulation comprising said stem cells; and optionally (iii) enrichingsaid stem cells or recovering said stem cells from the altered cellpopulation.
 45. A method according to claim 44 further comprisingrecommitting the cell having the appearance of one or more cell surfacemarkers of a stem cell into more cells having the appearance of one ormore cell surface markers of differentiated cells.
 46. A cell having theappearance of one or more cell surface markers of a stem cell producedaccording to the method of claim
 44. 47. A cell having the appearance ofone or more surface markers of differentiated cells produced accordingto the method of claim
 45. 48. A method for identifying a substancecapable of retrodifferentiating a committed/differentiated cell to anundifferentiated cell, which method comprises contacting a population ofcells comprising committed cells with a candidate substance anddetermining whether there is an increase in the relative numbers ofundifferentiated cells in said cell population.
 49. The method of claim48 wherein the committed cell is a hemopoietic cell.
 50. The method ofclaim 49 wherein the hemopoietic cell is selected from CFC-T cells,CFC-B cells, CFC-Eosin cells, CFC-Bas cells, CFC-GM cells, CFC-MEGcells, BFC-E cells, CFC-E cells, T cells and B cells.
 51. The method ofclaim 49 wherein the hemopoietic cell is a B cell.
 52. The method ofclaim 1 wherein said undifferentiated cell is optionally isolated and/orpurified.
 53. The method of claim 30 wherein said undifferentiated cellis optionally isolated and/or purified.
 54. The method of claim 31wherein said undifferentiated cell is optionally isolated and/orpurified.
 55. The method of claim 34 wherein said cell of anotherlineage is optionally isolated and/or purified.
 56. The method of claim36 wherein said T cell is optionally isolated and/or purified.
 57. Themethod of claim 37 wherein said myeloid cell is optionally isolatedand/or purified.
 58. The method of claim 38 wherein said stem cell isoptionally isolated and/or purified.
 59. The method of claim 39 whereinsaid cell with stem cell surface marker is optionally isolated and/orpurified.
 60. The method of claim 43 wherein said cell with stem cellsurface marker is optionally isolated and/or purified.
 61. The method ofclaim 44 wherein said cell with stem cell surface marker is optionallyisolated and/or purified.